streptavidin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin
    Sperm DNA oxidation in aging Prdx6 −/− and WT mice. (A) Immunocytochemistry showing 8-OHdG labeling in mouse spermatozoa. Cauda epididymis spermatozoa were permeabilized with methanol and incubated overnight with the anti-8-OHdG antibody and then with the biotin-labeled anti-mouse antibody followed by <t>streptavidin</t> conjugated to Alexa Fluor 555. All pictures were taken at 1000× magnification using the same exposure time. No labeling was observed with secondary antibody alone (data not shown). (B) Percentage of spermatozoa showing DNA oxidation (8-OHdG labeling). # Means higher than the other age-matched group and ## means higher than the other age groups of WT males ( p
    Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 169 article reviews
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    streptavidin - by Bioz Stars, 2020-03
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    Images

    1) Product Images from "Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice"

    Article Title: Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice

    Journal: Redox Biology

    doi: 10.1016/j.redox.2015.02.004

    Sperm DNA oxidation in aging Prdx6 −/− and WT mice. (A) Immunocytochemistry showing 8-OHdG labeling in mouse spermatozoa. Cauda epididymis spermatozoa were permeabilized with methanol and incubated overnight with the anti-8-OHdG antibody and then with the biotin-labeled anti-mouse antibody followed by streptavidin conjugated to Alexa Fluor 555. All pictures were taken at 1000× magnification using the same exposure time. No labeling was observed with secondary antibody alone (data not shown). (B) Percentage of spermatozoa showing DNA oxidation (8-OHdG labeling). # Means higher than the other age-matched group and ## means higher than the other age groups of WT males ( p
    Figure Legend Snippet: Sperm DNA oxidation in aging Prdx6 −/− and WT mice. (A) Immunocytochemistry showing 8-OHdG labeling in mouse spermatozoa. Cauda epididymis spermatozoa were permeabilized with methanol and incubated overnight with the anti-8-OHdG antibody and then with the biotin-labeled anti-mouse antibody followed by streptavidin conjugated to Alexa Fluor 555. All pictures were taken at 1000× magnification using the same exposure time. No labeling was observed with secondary antibody alone (data not shown). (B) Percentage of spermatozoa showing DNA oxidation (8-OHdG labeling). # Means higher than the other age-matched group and ## means higher than the other age groups of WT males ( p

    Techniques Used: Mouse Assay, Immunocytochemistry, Labeling, Incubation

    2) Product Images from "A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions"

    Article Title: A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv069

    The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through biotin–streptavidin interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.
    Figure Legend Snippet: The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through biotin–streptavidin interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.

    Techniques Used: High Throughput Screening Assay, Labeling, Blocking Assay, Binding Assay, Protein Binding, Fluorescence

    3) Product Images from "Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit"

    Article Title: Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-10-76

    Functional characterization of the Sbh1p tm-domain mutants . A, Multicopy suppression of temperature-sensitivity of sbh1Δ sbh2Δ cells (H3232) by different mutant forms of SBH1 tm-domain. The sbh1Δ sbh2Δ cells were transformed with BIO-tagged SBH1 tm-domain mutants expressed from the ADH1 promoter in p426ADH and the growth of four independent transformants was monitored at 38 and 24°C. B, Lysates prepared from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p TM, mutants TM-1 to TM-7 or the empty vector were subjected to pull-down with streptavidin-coated magnetic beads. Beads and input samples were analyzed by Western blotting with anti-Sec61 antibodies or with HRP conjugated streptavidin to detect different versions of BIO-Sbh1p.
    Figure Legend Snippet: Functional characterization of the Sbh1p tm-domain mutants . A, Multicopy suppression of temperature-sensitivity of sbh1Δ sbh2Δ cells (H3232) by different mutant forms of SBH1 tm-domain. The sbh1Δ sbh2Δ cells were transformed with BIO-tagged SBH1 tm-domain mutants expressed from the ADH1 promoter in p426ADH and the growth of four independent transformants was monitored at 38 and 24°C. B, Lysates prepared from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p TM, mutants TM-1 to TM-7 or the empty vector were subjected to pull-down with streptavidin-coated magnetic beads. Beads and input samples were analyzed by Western blotting with anti-Sec61 antibodies or with HRP conjugated streptavidin to detect different versions of BIO-Sbh1p.

    Techniques Used: Functional Assay, Mutagenesis, Transformation Assay, Expressing, Plasmid Preparation, Magnetic Beads, Western Blot

    The P54S V57G Sbh1p tm-domain double mutant is unable to rescue loss of Sbh1p and Sbh2p . A, A wheel presentation of the P54S V57G Sbh1p tm-domain mutant. B, Growth of sbh1Δ sbh2Δ cells (H3232) transformed with plasmids encoding SBH1 tm-domain mutants with or without BIO-tag. C, UTA translocation assay on sbh1Δ sbh2Δ cells (H3543) transformed with plasmids encoding reporter proteins Suc2 23 , Sec2 277 , Dap2 300 , or the empty vector pRS314, or with plasmids encoding full length Sbh1p with P54S V57G mutations (Sbh1-FL(DM)), Sbh1p tm-domain with P54S (Sbh1-TM(SM1)), Sbh1p tm-domain with V57G (Sbh1-TM(SM2)) or Sbh1p tm-domain with P54S V57G (Sbh1-TM(DM)) or the empty plasmid. The growth of transformants was tested on SCD-Trp-Leu or on SCD-Trp-Leu-Ura plates to score for translocation of the Ura3p containing reporters. D, Western blot analysis with anti-HA (for Rtn1p-HA), Sec61p antibodies or with HRP conjugated streptavidin (for versions of BIO-Sbh1p) of pull-downs from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p(P54S V57G), Sbh1p TM(P54S), Sbh1p TM(V57G), Sbh1p TM(P54S V57G) or an empty plasmid p426ADH.
    Figure Legend Snippet: The P54S V57G Sbh1p tm-domain double mutant is unable to rescue loss of Sbh1p and Sbh2p . A, A wheel presentation of the P54S V57G Sbh1p tm-domain mutant. B, Growth of sbh1Δ sbh2Δ cells (H3232) transformed with plasmids encoding SBH1 tm-domain mutants with or without BIO-tag. C, UTA translocation assay on sbh1Δ sbh2Δ cells (H3543) transformed with plasmids encoding reporter proteins Suc2 23 , Sec2 277 , Dap2 300 , or the empty vector pRS314, or with plasmids encoding full length Sbh1p with P54S V57G mutations (Sbh1-FL(DM)), Sbh1p tm-domain with P54S (Sbh1-TM(SM1)), Sbh1p tm-domain with V57G (Sbh1-TM(SM2)) or Sbh1p tm-domain with P54S V57G (Sbh1-TM(DM)) or the empty plasmid. The growth of transformants was tested on SCD-Trp-Leu or on SCD-Trp-Leu-Ura plates to score for translocation of the Ura3p containing reporters. D, Western blot analysis with anti-HA (for Rtn1p-HA), Sec61p antibodies or with HRP conjugated streptavidin (for versions of BIO-Sbh1p) of pull-downs from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p(P54S V57G), Sbh1p TM(P54S), Sbh1p TM(V57G), Sbh1p TM(P54S V57G) or an empty plasmid p426ADH.

    Techniques Used: Mutagenesis, Transformation Assay, Translocation Assay, Plasmid Preparation, Western Blot, Expressing

    Sbh1p co-immunoprecipitates with S. cerevisiae reticulon-homologues Rtn1p, Rtn2p and Yop1p . A, The sbh1Δ RTN1-3XHA RTN2-9xmyc cells (H3723) or in B, sbh1Δ RTN1-3XHA YOP1-9xmyc cells (H3722) were transformed with YEpBIO-SBH1, YEpBIO-SBH1-TM, or an empty plasmid p426ADH. Lysates were prepared and subjected to pull-down with streptavidin-conjugated magnetic beads followed by SDS-PAGE and Western blotting. Proteins were detected with HRP-conjugated streptavidin and antibodies to Sec61p, myc and HA.
    Figure Legend Snippet: Sbh1p co-immunoprecipitates with S. cerevisiae reticulon-homologues Rtn1p, Rtn2p and Yop1p . A, The sbh1Δ RTN1-3XHA RTN2-9xmyc cells (H3723) or in B, sbh1Δ RTN1-3XHA YOP1-9xmyc cells (H3722) were transformed with YEpBIO-SBH1, YEpBIO-SBH1-TM, or an empty plasmid p426ADH. Lysates were prepared and subjected to pull-down with streptavidin-conjugated magnetic beads followed by SDS-PAGE and Western blotting. Proteins were detected with HRP-conjugated streptavidin and antibodies to Sec61p, myc and HA.

    Techniques Used: Transformation Assay, Plasmid Preparation, Magnetic Beads, SDS Page, Western Blot

    4) Product Images from "Quantifying cell-generated mechanical forces within living embryonic tissues"

    Article Title: Quantifying cell-generated mechanical forces within living embryonic tissues

    Journal: Nature methods

    doi: 10.1038/nmeth.2761

    Oil microdroplets as force transducers ( a ) Sketch of isolated spherical oil droplets in solution (left) and a droplet embedded in-between the cells forming an embryonic tissue (right); the deformation of the droplet is a consequence of local cellular forces. ( b ) Confocal section of an isolated fluorocarbon oil droplet coated as described in the main text. Droplet surface is fluorescently labeled with Cy5-streptavidin. Bar, 10 µm. ( c ) Sketch of the interface between fluorocarbon oil and surrounding medium, indicating the different molecules involved in the coating (functionalization) of the droplets. ( d ) Sketch of fluorocarbon-hydrocarbon (Krytox-Dodecylamine) diblocks used to vary the interfacial tension and ( e ) surfactant molecules (DSPE-PEG-biotin) used to stabilize and control the surface properties of the droplet.
    Figure Legend Snippet: Oil microdroplets as force transducers ( a ) Sketch of isolated spherical oil droplets in solution (left) and a droplet embedded in-between the cells forming an embryonic tissue (right); the deformation of the droplet is a consequence of local cellular forces. ( b ) Confocal section of an isolated fluorocarbon oil droplet coated as described in the main text. Droplet surface is fluorescently labeled with Cy5-streptavidin. Bar, 10 µm. ( c ) Sketch of the interface between fluorocarbon oil and surrounding medium, indicating the different molecules involved in the coating (functionalization) of the droplets. ( d ) Sketch of fluorocarbon-hydrocarbon (Krytox-Dodecylamine) diblocks used to vary the interfacial tension and ( e ) surfactant molecules (DSPE-PEG-biotin) used to stabilize and control the surface properties of the droplet.

    Techniques Used: Isolation, Labeling

    5) Product Images from "B cell antigen extraction is regulated by physical properties of antigen-presenting cells"

    Article Title: B cell antigen extraction is regulated by physical properties of antigen-presenting cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201607064

    B cells change the antigen extraction mechanism depending on the physical properties of antigen-presenting substrates. (A) Schematic of the mechanism sensor. (B) Representative total internal reflection fluorescence images of the contact areas between B1-8 B cells and the mechanism (mech.) sensor (presenting NIP 10 antigen) tethered to PEG-coated glass, PLB, or PMS substrates. BF, bright field. (C) Side-view reconstructions of B220-stained B1-8 B cells that have extracted the mechanism sensor from each substrate. Note that the substrate surfaces are not clearly visible because of the deconvolution procedure for the image display, which removes smooth, unclustered signal from the images. Therefore, we included blue arrows to mark the positions of the substrates. (D) Quantification of Atto647N/Atto550 fluorescence intensity ratios in antigen (Ag) clusters internalized from PEG, PLB, and PMS substrates. (E) Quantification of total B cell antigen extraction (Atto550 fluorescence intensity) from each substrate (mean and SEM; cell numbers are shown in parentheses above each bar). (F) Quantification of streptavidin extracted, calculated as antigen cluster AF405/Atto550 intensity ratios. Error bars in D and F represent mean and SEM for the cluster numbers shown in parentheses above each bar. Data are from one experiment representative of three independent experiments. **, P
    Figure Legend Snippet: B cells change the antigen extraction mechanism depending on the physical properties of antigen-presenting substrates. (A) Schematic of the mechanism sensor. (B) Representative total internal reflection fluorescence images of the contact areas between B1-8 B cells and the mechanism (mech.) sensor (presenting NIP 10 antigen) tethered to PEG-coated glass, PLB, or PMS substrates. BF, bright field. (C) Side-view reconstructions of B220-stained B1-8 B cells that have extracted the mechanism sensor from each substrate. Note that the substrate surfaces are not clearly visible because of the deconvolution procedure for the image display, which removes smooth, unclustered signal from the images. Therefore, we included blue arrows to mark the positions of the substrates. (D) Quantification of Atto647N/Atto550 fluorescence intensity ratios in antigen (Ag) clusters internalized from PEG, PLB, and PMS substrates. (E) Quantification of total B cell antigen extraction (Atto550 fluorescence intensity) from each substrate (mean and SEM; cell numbers are shown in parentheses above each bar). (F) Quantification of streptavidin extracted, calculated as antigen cluster AF405/Atto550 intensity ratios. Error bars in D and F represent mean and SEM for the cluster numbers shown in parentheses above each bar. Data are from one experiment representative of three independent experiments. **, P

    Techniques Used: Fluorescence, Staining

    Mechanical force is the dominant antigen extraction mechanism. (A) Schematic of the DNA-based tension sensor. Ag, antigen. (B) Side-view reconstruction of a B cell that has unfolded the tension sensor to extract the antigen (NIP 10 ) and upper handle (Atto647N) from a PLB, leaving the lower handle (Atto550) of the sensor behind. The blue arrows indicate the position of the substrate. Bar, 5 µm (C) Antigen (NIP 10 ) extracted through enzymatic degradation when 0% or 0.1% of the antigen is attached to the tension sensor. Error bars represent mean Atto550 fluorescence intensity in extracted antigen clusters and SEM for the cell numbers shown in parentheses above each bar. Data are from one experiment representative of two independent experiments. f.u., fluorescence units. (D) LAMP-1 + vesicle recruitment to the plasma membrane calculated as the ratio of synaptic to total cell LAMP-1 intensity for B1-8 B cells binding NIP 10 antigen tethered to the mechanism (mech) sensor, tension sensor, or a 1:1 sensor mixture on PLBs. (E) LAMP-1 + vesicle recruitment to the plasma membrane for B1-8 B cells that either did (+) or did not (−) internalize NIP 10 antigen from the tension sensor on PLBs. In D and E, data are mean and SEM for the cell numbers indicated in parentheses above each bar, and are from one experiment representative of two independent experiments. (F) Side-view reconstructions showing antigen and LAMP-1 + vesicles in naive B cells binding antigen (anti-Igκ) anchored directly via biotin–streptavidin–biotin linkers to PMSs or PLBs. Bars, 5 µm. (G) Time course of LAMP-1 + vesicle recruitment to the plasma membrane on PLBs and PMSs (mean and SEM; cell numbers are shown in parentheses adjacent to the markers). Data are from one experiment representative of two independent experiments. ****, P
    Figure Legend Snippet: Mechanical force is the dominant antigen extraction mechanism. (A) Schematic of the DNA-based tension sensor. Ag, antigen. (B) Side-view reconstruction of a B cell that has unfolded the tension sensor to extract the antigen (NIP 10 ) and upper handle (Atto647N) from a PLB, leaving the lower handle (Atto550) of the sensor behind. The blue arrows indicate the position of the substrate. Bar, 5 µm (C) Antigen (NIP 10 ) extracted through enzymatic degradation when 0% or 0.1% of the antigen is attached to the tension sensor. Error bars represent mean Atto550 fluorescence intensity in extracted antigen clusters and SEM for the cell numbers shown in parentheses above each bar. Data are from one experiment representative of two independent experiments. f.u., fluorescence units. (D) LAMP-1 + vesicle recruitment to the plasma membrane calculated as the ratio of synaptic to total cell LAMP-1 intensity for B1-8 B cells binding NIP 10 antigen tethered to the mechanism (mech) sensor, tension sensor, or a 1:1 sensor mixture on PLBs. (E) LAMP-1 + vesicle recruitment to the plasma membrane for B1-8 B cells that either did (+) or did not (−) internalize NIP 10 antigen from the tension sensor on PLBs. In D and E, data are mean and SEM for the cell numbers indicated in parentheses above each bar, and are from one experiment representative of two independent experiments. (F) Side-view reconstructions showing antigen and LAMP-1 + vesicles in naive B cells binding antigen (anti-Igκ) anchored directly via biotin–streptavidin–biotin linkers to PMSs or PLBs. Bars, 5 µm. (G) Time course of LAMP-1 + vesicle recruitment to the plasma membrane on PLBs and PMSs (mean and SEM; cell numbers are shown in parentheses adjacent to the markers). Data are from one experiment representative of two independent experiments. ****, P

    Techniques Used: Fluorescence, Binding Assay

    Substrate flexibility, antigen tethering strength, and antigen affinity regulate B cell antigen extraction. (A) Side-view reconstructions of B1-8 B cells that have extracted antigen (NIP 10 ) from the tension sensor on stiff (PLB) and flexible (PMS) substrates. (B) Extraction of the tension sensor’s lower handle or streptavidin by B1-8 B cells from PLBs or PMSs. Data are mean fluorescence intensities in extracted antigen clusters (Atto550, lower handle; AF405, streptavidin) normalized to Atto647N fluorescence ± SEM. Cluster numbers are shown above the bars. (C) Extraction of NIP 10 antigen (Ag) by B1-8 B cells from tension sensors providing a weak or strong tether from PLBs or PMSs. Graphs represent mean extracted antigen percent per cell and SEM. Data are from one experiment representative of three independent experiments. (D and E) Extraction of NIP 1 and NP 1 antigens by B1-8 B cells from weak and strong tethers from PLBs (D) or PMSs (E). Data are mean and SEM from one experiment representative of three independent experiments. (F) Ratio of NIP 1 to NP 1 antigens extracted by B1-8 B cells from weak and strong tethers from PLBs and PMSs. Data are mean and SEM from three experiments on each substrate. In C–E, cell numbers are shown in parentheses above each bar. ****, P
    Figure Legend Snippet: Substrate flexibility, antigen tethering strength, and antigen affinity regulate B cell antigen extraction. (A) Side-view reconstructions of B1-8 B cells that have extracted antigen (NIP 10 ) from the tension sensor on stiff (PLB) and flexible (PMS) substrates. (B) Extraction of the tension sensor’s lower handle or streptavidin by B1-8 B cells from PLBs or PMSs. Data are mean fluorescence intensities in extracted antigen clusters (Atto550, lower handle; AF405, streptavidin) normalized to Atto647N fluorescence ± SEM. Cluster numbers are shown above the bars. (C) Extraction of NIP 10 antigen (Ag) by B1-8 B cells from tension sensors providing a weak or strong tether from PLBs or PMSs. Graphs represent mean extracted antigen percent per cell and SEM. Data are from one experiment representative of three independent experiments. (D and E) Extraction of NIP 1 and NP 1 antigens by B1-8 B cells from weak and strong tethers from PLBs (D) or PMSs (E). Data are mean and SEM from one experiment representative of three independent experiments. (F) Ratio of NIP 1 to NP 1 antigens extracted by B1-8 B cells from weak and strong tethers from PLBs and PMSs. Data are mean and SEM from three experiments on each substrate. In C–E, cell numbers are shown in parentheses above each bar. ****, P

    Techniques Used: Fluorescence

    6) Product Images from "lncRNA SLC7A11-AS1 Promotes Chemoresistance by Blocking SCFβ-TRCP-Mediated Degradation of NRF2 in Pancreatic Cancer"

    Article Title: lncRNA SLC7A11-AS1 Promotes Chemoresistance by Blocking SCFβ-TRCP-Mediated Degradation of NRF2 in Pancreatic Cancer

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.11.035

    SLC7A11-AS1 Prevents β-TRCP1-Mediated Ubiquitination and Degradation of NRF2 (A and B) Subcellular distribution of SLC7A11-AS1 was detected by qRT-PCR in (A) BxPC-3-Gem and (B) PANC-1 cells. U99 and MT-RNR1 were as nuclear and cytoplasmic marker, respectively (n = 3). (C) The immunofluorescence staining of SLC7A11-AS1 (red), β-TRCP1 (green), and DAPI (blue) in BxPC-3-Gem and PANC-1 cells. (D) Nuclear extracts from BxPC-3-Gem cells were incubated with biotinylated sense and antisense SLC7A11-AS1 generated in vitro , and proteins were precipitated with streptavidin beads and subjected to immunoblotting (IB) analysis with anti-TRCP1 antibody. (E) A schematic diagram of full-length SLC7A11-AS1 and its series of truncates. (F) Nuclear extracts from BxPC-3-Gem cells were incubated with biotinylated SLC7A11-AS1 truncates and antisense SLC7A11-AS1 generated in vitro , and proteins were precipitated with streptavidin beads and subjected to IB analysis with anti-TRCP1 antibody. (G) A schematic diagram of β-TRCP1 and its truncates. (H) RIP assay analysis of the interaction of β-TRCP1 and its truncates (β-TRCP1-N, β-TRCP1-N-ΔF-box, and β-TRCP1-C) with SLC7A11-AS1 in PANC-1 cells. Whole-cell expression (input) of proteins was detected by IB with indicated antibodies (left). The asterisks indicate β-TRCP1 and its truncates bands. The immunoprecipitated SLC7A11-AS1 by using anti-HA antibody was measured by qRT-PCR and represented as a fraction of input RNA (% input) prior to immunoprecipitation (right) (n = 3). (I) Effect of SLC7A11-AS1 on β-TRCP1-mediated ubiquitination of NRF2. 293T cells co-transfected with indicated plasmids were treated with MG132 (1 μM) for 12 h and subjected to immunoprecipitation (IP) with the anti-Myc antibody, followed by IB with anti-HA antibody. Whole-cell expression (input) of proteins was detected by IB with anti-FLAG or anti-GAPDH antibodies. (J) SLC7A11-AS1 prevents β-catenin proteasomal degradation. Western blot (left) analysis of nuclear β-catenin in SLC7A11-AS1 -knockdown and control PANC-1 cells treated with or without MG132 (5 μM) for 24 h. Quantification of β-catenin protein (right) was normalized to the loading control (Lamin) and expressed relative to sh-Ctrl without MG132 (n = 3). **p
    Figure Legend Snippet: SLC7A11-AS1 Prevents β-TRCP1-Mediated Ubiquitination and Degradation of NRF2 (A and B) Subcellular distribution of SLC7A11-AS1 was detected by qRT-PCR in (A) BxPC-3-Gem and (B) PANC-1 cells. U99 and MT-RNR1 were as nuclear and cytoplasmic marker, respectively (n = 3). (C) The immunofluorescence staining of SLC7A11-AS1 (red), β-TRCP1 (green), and DAPI (blue) in BxPC-3-Gem and PANC-1 cells. (D) Nuclear extracts from BxPC-3-Gem cells were incubated with biotinylated sense and antisense SLC7A11-AS1 generated in vitro , and proteins were precipitated with streptavidin beads and subjected to immunoblotting (IB) analysis with anti-TRCP1 antibody. (E) A schematic diagram of full-length SLC7A11-AS1 and its series of truncates. (F) Nuclear extracts from BxPC-3-Gem cells were incubated with biotinylated SLC7A11-AS1 truncates and antisense SLC7A11-AS1 generated in vitro , and proteins were precipitated with streptavidin beads and subjected to IB analysis with anti-TRCP1 antibody. (G) A schematic diagram of β-TRCP1 and its truncates. (H) RIP assay analysis of the interaction of β-TRCP1 and its truncates (β-TRCP1-N, β-TRCP1-N-ΔF-box, and β-TRCP1-C) with SLC7A11-AS1 in PANC-1 cells. Whole-cell expression (input) of proteins was detected by IB with indicated antibodies (left). The asterisks indicate β-TRCP1 and its truncates bands. The immunoprecipitated SLC7A11-AS1 by using anti-HA antibody was measured by qRT-PCR and represented as a fraction of input RNA (% input) prior to immunoprecipitation (right) (n = 3). (I) Effect of SLC7A11-AS1 on β-TRCP1-mediated ubiquitination of NRF2. 293T cells co-transfected with indicated plasmids were treated with MG132 (1 μM) for 12 h and subjected to immunoprecipitation (IP) with the anti-Myc antibody, followed by IB with anti-HA antibody. Whole-cell expression (input) of proteins was detected by IB with anti-FLAG or anti-GAPDH antibodies. (J) SLC7A11-AS1 prevents β-catenin proteasomal degradation. Western blot (left) analysis of nuclear β-catenin in SLC7A11-AS1 -knockdown and control PANC-1 cells treated with or without MG132 (5 μM) for 24 h. Quantification of β-catenin protein (right) was normalized to the loading control (Lamin) and expressed relative to sh-Ctrl without MG132 (n = 3). **p

    Techniques Used: Quantitative RT-PCR, Marker, Immunofluorescence, Staining, Incubation, Generated, In Vitro, Expressing, Immunoprecipitation, Transfection, Western Blot

    7) Product Images from "Molecular Occupancy of Nanodot Arrays"

    Article Title: Molecular Occupancy of Nanodot Arrays

    Journal: ACS nano

    doi: 10.1021/acsnano.5b07425

    (a) Molecular model showing the distance distribution from the fluorophores (approximated by the nitrogen atoms on primary amine of lysine) on streptavidin to the nanodot surface. (b) Schematic curves of quenching efficiency vs separation distance for
    Figure Legend Snippet: (a) Molecular model showing the distance distribution from the fluorophores (approximated by the nitrogen atoms on primary amine of lysine) on streptavidin to the nanodot surface. (b) Schematic curves of quenching efficiency vs separation distance for

    Techniques Used:

    (a) Schematic diagram of the bifunctional nanoarray surface with a live T-cell. (b) Fluorescence intensity of both streptavidin-647 and UCHT1 Fab′-568 for hexagonal arrays with various spacings. Normalized pY intensity after 5 min stimulation:
    Figure Legend Snippet: (a) Schematic diagram of the bifunctional nanoarray surface with a live T-cell. (b) Fluorescence intensity of both streptavidin-647 and UCHT1 Fab′-568 for hexagonal arrays with various spacings. Normalized pY intensity after 5 min stimulation:

    Techniques Used: Fluorescence

    Schematic diagram of the metallic nanodot array: (a) annealing, (b) functionalization, (c) excitation and photobleaching of the fluorophores labeled on a streptavidin (only the SAM in the cross-section is shown for clarity). (d) SEM image of the NIL mold
    Figure Legend Snippet: Schematic diagram of the metallic nanodot array: (a) annealing, (b) functionalization, (c) excitation and photobleaching of the fluorophores labeled on a streptavidin (only the SAM in the cross-section is shown for clarity). (d) SEM image of the NIL mold

    Techniques Used: Labeling

    8) Product Images from "Characterization of the evanescent field profile and bound mass sensitivity of a label-free silicon photonic microring resonator biosensing platform"

    Article Title: Characterization of the evanescent field profile and bound mass sensitivity of a label-free silicon photonic microring resonator biosensing platform

    Journal: Biosensors & bioelectronics

    doi: 10.1016/j.bios.2010.07.010

    (A) Schematic showing the APTES-modified silicon surface coated with a layer of NHS-biotin followed by a layer of 125 I-labeled streptavidin. (B) Real time data showing binding of 125 I-labeled streptavidin to a biotin-functionalized surface. After rinsing the surface with buffer, the average net shift (relative shift after streptavidin minus relative shift before streptavidin) is measured for multiple rings. (C) Phosphorimage showing the relative intensity of 125 I-streptavidin standards compared with that bound on the U-shaped area of the chip to which flow was directed by microfluidics. The red rectangle highlights the region of interest directly over the rings on the chip where the radioactivity intensity was measured. In this selected area, the cladding layer was etched off exposing silicon oxide over the entire surface. (D) Calibration curve relating the mass of spotted protein standards (black dots) from (C) to the phosphorimager intensity obtained. The blue square represents the signal arising from the selected area (box in C).
    Figure Legend Snippet: (A) Schematic showing the APTES-modified silicon surface coated with a layer of NHS-biotin followed by a layer of 125 I-labeled streptavidin. (B) Real time data showing binding of 125 I-labeled streptavidin to a biotin-functionalized surface. After rinsing the surface with buffer, the average net shift (relative shift after streptavidin minus relative shift before streptavidin) is measured for multiple rings. (C) Phosphorimage showing the relative intensity of 125 I-streptavidin standards compared with that bound on the U-shaped area of the chip to which flow was directed by microfluidics. The red rectangle highlights the region of interest directly over the rings on the chip where the radioactivity intensity was measured. In this selected area, the cladding layer was etched off exposing silicon oxide over the entire surface. (D) Calibration curve relating the mass of spotted protein standards (black dots) from (C) to the phosphorimager intensity obtained. The blue square represents the signal arising from the selected area (box in C).

    Techniques Used: Modification, Labeling, Binding Assay, Chromatin Immunoprecipitation, Flow Cytometry, Radioactivity

    (A) Schematic showing the layer-by-layer addition of biotinylated antibody (Ab) and streptavidin (SA) to the ring resonator surface. Layer 1 shows covalent attachment of 4FB-modified, biotinylated antibody to the HyNic-silane functionalized silicon surface. We hypothesize that initial antibody functionalization yields incomplete surface coverage, which is followed by an annealing process that initially results in non-linear layer growth as vacancies are filled. Layers 2 and 3 show subsequent addition of streptavidin and biotinylated antibody, respectively. Extended multilayers are formed by alternating deposition of streptavidin and biotinylated antibody. Though each antibody is functionalized with ∼10 biotins, fewer are shown for clarity. (B) Real time data showing the relative shift in resonance frequency (Δ pm) upon addition of SA and Ab layers. Inset shows six layer-by-layer depositions at higher resolution. (C) Plot showing the cumulative relative shift per antibody or streptavidin layer (each individual addition of streptavidin or antibody is counted as a single layer). The exponential fit shown in the plot indicates the decay rate starting from layer 16. Error bars represent the standard deviation for n = 4 rings.
    Figure Legend Snippet: (A) Schematic showing the layer-by-layer addition of biotinylated antibody (Ab) and streptavidin (SA) to the ring resonator surface. Layer 1 shows covalent attachment of 4FB-modified, biotinylated antibody to the HyNic-silane functionalized silicon surface. We hypothesize that initial antibody functionalization yields incomplete surface coverage, which is followed by an annealing process that initially results in non-linear layer growth as vacancies are filled. Layers 2 and 3 show subsequent addition of streptavidin and biotinylated antibody, respectively. Extended multilayers are formed by alternating deposition of streptavidin and biotinylated antibody. Though each antibody is functionalized with ∼10 biotins, fewer are shown for clarity. (B) Real time data showing the relative shift in resonance frequency (Δ pm) upon addition of SA and Ab layers. Inset shows six layer-by-layer depositions at higher resolution. (C) Plot showing the cumulative relative shift per antibody or streptavidin layer (each individual addition of streptavidin or antibody is counted as a single layer). The exponential fit shown in the plot indicates the decay rate starting from layer 16. Error bars represent the standard deviation for n = 4 rings.

    Techniques Used: Modification, Standard Deviation

    Plot displaying the relative differential shift per antibody/streptavidin bilayer. Notably, the signal increases initially to a maximum shift of ∼250 pm at bilayer 8. Beyond this point the signal from subsequent layers decreases in an exponential fashion as they are deposited further away from the surface where there is a lower evanescent field intensity. Error bars represent the standard deviation for n = 4 rings.
    Figure Legend Snippet: Plot displaying the relative differential shift per antibody/streptavidin bilayer. Notably, the signal increases initially to a maximum shift of ∼250 pm at bilayer 8. Beyond this point the signal from subsequent layers decreases in an exponential fashion as they are deposited further away from the surface where there is a lower evanescent field intensity. Error bars represent the standard deviation for n = 4 rings.

    Techniques Used: Standard Deviation

    9) Product Images from "Deformations in Actin Comets from Rocketing Beads"

    Article Title: Deformations in Actin Comets from Rocketing Beads

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.106.088054

    Bead velocity depends on bead radius and on the presence of cross-linkers. Beads were incubated in a medium containing 8.1 μ M F-actin (10% labeled with Alexa Fluor 594), 0.1 μ M Arp2/3, 3 μ M ADF/cofilin, 1 μ M profilin, and various concentrations of gelsolin. ( a ) The average velocity as a function of the bead radius for gelsolin concentrations of 0.35 μ M ( solid diamonds ), 0.54 μ M ( shaded triangles ), and 0.73 μ M ( squares ). The data for 0.35 μ M is fitted with v = 0.25/ R ( χ 2 = 0.0019). Other continuous lines are to guide the eyes. ( b ) The average velocity of 1.5- μ m radius beads as a function of the concentration of streptavidin in the presence of 2.5 μ M biotinylated actin (30% of total actin) for gelsolin concentrations of 0.35 μ M ( solid diamonds ), 0.54 μ M ( shaded triangles ), 0.73 μ M ( squares ), and 0.92 μ M ( light shaded down - triangles ). ( c ) The effect of the cross-linkers filamin ( squares ), α -actinin ( light shaded down - triangles ), and fascin ( shaded diamonds ) on the average velocity of 1.5- μ m radius beads for gelsolin concentrations of 0.35 μ M ( open symbols ) and 0.9 μ M ( solid symbols ).
    Figure Legend Snippet: Bead velocity depends on bead radius and on the presence of cross-linkers. Beads were incubated in a medium containing 8.1 μ M F-actin (10% labeled with Alexa Fluor 594), 0.1 μ M Arp2/3, 3 μ M ADF/cofilin, 1 μ M profilin, and various concentrations of gelsolin. ( a ) The average velocity as a function of the bead radius for gelsolin concentrations of 0.35 μ M ( solid diamonds ), 0.54 μ M ( shaded triangles ), and 0.73 μ M ( squares ). The data for 0.35 μ M is fitted with v = 0.25/ R ( χ 2 = 0.0019). Other continuous lines are to guide the eyes. ( b ) The average velocity of 1.5- μ m radius beads as a function of the concentration of streptavidin in the presence of 2.5 μ M biotinylated actin (30% of total actin) for gelsolin concentrations of 0.35 μ M ( solid diamonds ), 0.54 μ M ( shaded triangles ), 0.73 μ M ( squares ), and 0.92 μ M ( light shaded down - triangles ). ( c ) The effect of the cross-linkers filamin ( squares ), α -actinin ( light shaded down - triangles ), and fascin ( shaded diamonds ) on the average velocity of 1.5- μ m radius beads for gelsolin concentrations of 0.35 μ M ( open symbols ) and 0.9 μ M ( solid symbols ).

    Techniques Used: Incubation, Labeling, Concentration Assay

    10) Product Images from "The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme"

    Article Title: The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001933

    Identification of Nck1 binding to cytoplasmic CE. (A) HEK293 cells were co-transfected with plasmids expressing HA-Nck1 and bio-cCE (lanes 1 and 4), bio-cCEΔ25C (lanes 2 and 5), and bio-cCEΔpro in which the five C-terminal prolines shown in Figure 1A were deleted (bio-cCEΔpro, lanes 3 and 6). Protein recovered on streptavidin beads was analyzed by Western blotting with anti-Myc (cCE) and anti-HA (Nck1) antibodies (upper two panels). In the lower two panels the bound complexes were assayed as in Figure 1 for guanylylation and capping activity. The quantified capping activity (mean ± standard deviation, n = 3) for capping assay is shown beneath that autoradiogram. The same results were obtained for each of the modified forms of CE, with p -value
    Figure Legend Snippet: Identification of Nck1 binding to cytoplasmic CE. (A) HEK293 cells were co-transfected with plasmids expressing HA-Nck1 and bio-cCE (lanes 1 and 4), bio-cCEΔ25C (lanes 2 and 5), and bio-cCEΔpro in which the five C-terminal prolines shown in Figure 1A were deleted (bio-cCEΔpro, lanes 3 and 6). Protein recovered on streptavidin beads was analyzed by Western blotting with anti-Myc (cCE) and anti-HA (Nck1) antibodies (upper two panels). In the lower two panels the bound complexes were assayed as in Figure 1 for guanylylation and capping activity. The quantified capping activity (mean ± standard deviation, n = 3) for capping assay is shown beneath that autoradiogram. The same results were obtained for each of the modified forms of CE, with p -value

    Techniques Used: Binding Assay, Transfection, Expressing, Western Blot, Activity Assay, Standard Deviation, Modification

    The cytoplasmic capping complex assembles on Nck1. (A) Gst and Gst-Nck1 were expressed in E. coli and protein purified on glutathione agarose was analyzed by SDS-PAGE stained with Coomassie Blue (left panel). The middle panel is a Western blot using HRP-streptavidin of cytoplasmic extracts of cells that were transfected with plasmids expressing bio-cCE or MS2-bio. Gst- or Gst-Nck1 bound glutathione beads were added to each of these extracts (right panels) and bound proteins were analyzed by Western blotting with HRP-streptavidin (top panel), for guanylylation activity (middle panel), and for capping activity (bottom panel). The single band in guanylylation assay of MS2-bio expressing cells is endogenous CE, the two bands in bio-cCE expressing cells correspond to endogenous CE and bio-cCE. (B) 293 cells were transfected with Nck1 siRNA or a scrambled control and plasmid expressing bio-cCE. The effectiveness of Nck1 knockdown was determined by Western blotting with anti-Nck1 and anti-GAPDH antibodies, and the expression of bio-cCE and its recovery on streptavidin beads was monitored by Western blotting with anti-Myc antibody. The recovery of Nck1 with bio-cCE was determined by Western blotting with anti-Nck1 antibody (upper panels). In the bottom two panels the recovered complexes were assayed for recovery of 5′-kinase activity and capping activity, normalized to bio-cCE recovery, with the mean ± standard deviation ( n = 3) shown beneath each autoradiogram. For each of these p -value was
    Figure Legend Snippet: The cytoplasmic capping complex assembles on Nck1. (A) Gst and Gst-Nck1 were expressed in E. coli and protein purified on glutathione agarose was analyzed by SDS-PAGE stained with Coomassie Blue (left panel). The middle panel is a Western blot using HRP-streptavidin of cytoplasmic extracts of cells that were transfected with plasmids expressing bio-cCE or MS2-bio. Gst- or Gst-Nck1 bound glutathione beads were added to each of these extracts (right panels) and bound proteins were analyzed by Western blotting with HRP-streptavidin (top panel), for guanylylation activity (middle panel), and for capping activity (bottom panel). The single band in guanylylation assay of MS2-bio expressing cells is endogenous CE, the two bands in bio-cCE expressing cells correspond to endogenous CE and bio-cCE. (B) 293 cells were transfected with Nck1 siRNA or a scrambled control and plasmid expressing bio-cCE. The effectiveness of Nck1 knockdown was determined by Western blotting with anti-Nck1 and anti-GAPDH antibodies, and the expression of bio-cCE and its recovery on streptavidin beads was monitored by Western blotting with anti-Myc antibody. The recovery of Nck1 with bio-cCE was determined by Western blotting with anti-Nck1 antibody (upper panels). In the bottom two panels the recovered complexes were assayed for recovery of 5′-kinase activity and capping activity, normalized to bio-cCE recovery, with the mean ± standard deviation ( n = 3) shown beneath each autoradiogram. For each of these p -value was

    Techniques Used: Purification, SDS Page, Staining, Western Blot, Transfection, Expressing, Activity Assay, Plasmid Preparation, Standard Deviation

    Identification of Nck1 as a component of the cytoplasmic capping complex. (A) Cytoplasmic extract from cells expressing bio-cCE was separated on a 10%–50% glycerol gradient. Fractions containing each of these proteins were identified by Western blotting of input fractions with antibodies to the Myc tag on bio-cCE and to Nck1 (upper 2 panels). Streptavidin beads were used to recover bio-cCE from individual fractions and bound proteins were again analyzed by Western blotting with anti-Myc and anti-Nck1 antibodies (lower two panels). (B) Cytoplasmic extract from non-transfected cells was separated on a calibrated Sephacryl S-200 column. Starting with the void volume individual fractions were collected and analyzed by Western blotting with anti-CE and anti-Nck1 antibodies. The missing CE band in fraction 3 was due to sample loss during loading. (C) The fractions indicated with a box at the bottom of (B) were pooled and immunoprecipitated with anti-Nck1 or control IgG. 20% of the immunoprecipitated sample was used for Western blotting with anti-Nck1 antibody and 70% of the immunoprecipitated sample was used for Western blotting with anti-CE antibody. (D) Cytoplasmic extract from non-transfected cells was immunoprecipitated with anti-CE antibody or control IgG, and the recovered proteins were analyzed by Western blotting with anti-Nck1 antibody.
    Figure Legend Snippet: Identification of Nck1 as a component of the cytoplasmic capping complex. (A) Cytoplasmic extract from cells expressing bio-cCE was separated on a 10%–50% glycerol gradient. Fractions containing each of these proteins were identified by Western blotting of input fractions with antibodies to the Myc tag on bio-cCE and to Nck1 (upper 2 panels). Streptavidin beads were used to recover bio-cCE from individual fractions and bound proteins were again analyzed by Western blotting with anti-Myc and anti-Nck1 antibodies (lower two panels). (B) Cytoplasmic extract from non-transfected cells was separated on a calibrated Sephacryl S-200 column. Starting with the void volume individual fractions were collected and analyzed by Western blotting with anti-CE and anti-Nck1 antibodies. The missing CE band in fraction 3 was due to sample loss during loading. (C) The fractions indicated with a box at the bottom of (B) were pooled and immunoprecipitated with anti-Nck1 or control IgG. 20% of the immunoprecipitated sample was used for Western blotting with anti-Nck1 antibody and 70% of the immunoprecipitated sample was used for Western blotting with anti-CE antibody. (D) Cytoplasmic extract from non-transfected cells was immunoprecipitated with anti-CE antibody or control IgG, and the recovered proteins were analyzed by Western blotting with anti-Nck1 antibody.

    Techniques Used: Expressing, Western Blot, Transfection, Immunoprecipitation

    The capping enzyme C-terminus participates in assembly of the cytoplasmic capping complex. (A) The organization of vertebrate CE is shown with the N-terminal triphosphatase domain indicated in white, the guanylyltransferase domain indicated in grey, and the proline-rich C-terminus indicated by the black oval. The proximity of the proline-rich sequence to the C-terminus is indicated by the sequence of the last 12 amino acids of human CE. (B) The forms of CE used to analyze the impact of C-terminal modifications is shown. In the nomenclature used here CE corresponds to full-length protein with an N-terminal Myc tag. CEΔ25C is the same protein missing the C-terminal 25 amino acids. This deletion includes the NLS. CEΔNLS+NES was described in Otsuka and colleagues [11] and consists of Myc-tagged CE with the NLS deleted (X), a C-terminal FLAG tag and the HIV Rev NES (black box). In bio-cCE the NLS is deleted and a sequence that is biotinylated in vivo is added upstream of Myc tag and Rev NES. In bio-cCEΔ25C the C-terminal 25 amino acids of this protein were deleted. cCE-bio is similar to CEΔNLS+NES except that the C-terminal FLAG tag is replaced with the biotinylation sequence. (C) The plasmids shown in (B) or a plasmid expressing Myc-tagged GFP were transfected into HEK293 cells. GFP or CE and its associated proteins were recovered with anti-Myc beads (CE, CEΔ25C, cCE-bio, and CEΔNLS+NES) or streptavidin beads (bio-cCE and bio-cCEΔ25C). The recovered protein was incubated with α-[ 32 P]GTP and analyzed for the covalent binding of [ 32 P]GMP (guanylylation) [11] , and for [ 32 P]GMP labeling of 5′-monophosphate RNA (capping). The products were separated by denaturing gel electrophoresis and visualized by autoradiography. The amount of capping activity relative to guanylylation activity is shown at the bottom of the figure, with results normalized to either CE or to bio-cCE as indicated by the vertical line. (D) The experiment in (C) was repeated with an additional assay for 5′-kinase activity. The vertical line in the Western blot and guanylylation assay is to indicate that CE and CEΔ25C were separated by empty lanes on each of these gels. In (C and D) the mean ± standard deviation ( n = 3) for recovered kinase and capping activity normalized to guanylylation activity is shown beneath each autoradiogram. In each case comparison to matching controls yielded p -value
    Figure Legend Snippet: The capping enzyme C-terminus participates in assembly of the cytoplasmic capping complex. (A) The organization of vertebrate CE is shown with the N-terminal triphosphatase domain indicated in white, the guanylyltransferase domain indicated in grey, and the proline-rich C-terminus indicated by the black oval. The proximity of the proline-rich sequence to the C-terminus is indicated by the sequence of the last 12 amino acids of human CE. (B) The forms of CE used to analyze the impact of C-terminal modifications is shown. In the nomenclature used here CE corresponds to full-length protein with an N-terminal Myc tag. CEΔ25C is the same protein missing the C-terminal 25 amino acids. This deletion includes the NLS. CEΔNLS+NES was described in Otsuka and colleagues [11] and consists of Myc-tagged CE with the NLS deleted (X), a C-terminal FLAG tag and the HIV Rev NES (black box). In bio-cCE the NLS is deleted and a sequence that is biotinylated in vivo is added upstream of Myc tag and Rev NES. In bio-cCEΔ25C the C-terminal 25 amino acids of this protein were deleted. cCE-bio is similar to CEΔNLS+NES except that the C-terminal FLAG tag is replaced with the biotinylation sequence. (C) The plasmids shown in (B) or a plasmid expressing Myc-tagged GFP were transfected into HEK293 cells. GFP or CE and its associated proteins were recovered with anti-Myc beads (CE, CEΔ25C, cCE-bio, and CEΔNLS+NES) or streptavidin beads (bio-cCE and bio-cCEΔ25C). The recovered protein was incubated with α-[ 32 P]GTP and analyzed for the covalent binding of [ 32 P]GMP (guanylylation) [11] , and for [ 32 P]GMP labeling of 5′-monophosphate RNA (capping). The products were separated by denaturing gel electrophoresis and visualized by autoradiography. The amount of capping activity relative to guanylylation activity is shown at the bottom of the figure, with results normalized to either CE or to bio-cCE as indicated by the vertical line. (D) The experiment in (C) was repeated with an additional assay for 5′-kinase activity. The vertical line in the Western blot and guanylylation assay is to indicate that CE and CEΔ25C were separated by empty lanes on each of these gels. In (C and D) the mean ± standard deviation ( n = 3) for recovered kinase and capping activity normalized to guanylylation activity is shown beneath each autoradiogram. In each case comparison to matching controls yielded p -value

    Techniques Used: Sequencing, FLAG-tag, In Vivo, Plasmid Preparation, Expressing, Transfection, Incubation, Binding Assay, Labeling, Nucleic Acid Electrophoresis, Autoradiography, Activity Assay, Western Blot, Standard Deviation

    A functional role for Nck1 in cap homeostasis. Triplicate cultures of U2OS cells were transfected with plasmids expressing HA-tagged wild-type Nck1, Nck1 mutated in the CE-binding domain (M3), or the 5′-kinase-binding domain (M2). Western blots showing overexpression of each of these proteins are in Figure S4 . The appearance of uncapped forms of transcripts in the “capping inhibited” pool (grey bars) was determined by their recovery on streptavidin beads after ligation of an RNA adapter and hybridization to a biotinylated antisense DNA oligonucleotide [13] . Each preparation contained an equal amount of uncapped human β-globin mRNA as an internal control and RNA recovered from M3 (A) and M2 (B) expressing cells was analyzed by qRT-PCR for four transcripts that accumulate uncapped forms in cells that are inhibited for cytoplasmic capping (DNAJB1, ILF2, MAPK1, RAB1A). The results are normalized to the signal from cells expressing wild-type Nck1. RNA from M3 (C) or M2 (D) expressing cells was also analyzed by qRT-PCR for three transcripts of the “uninduced” pool whose steady state levels are reduced when cytoplasmic capping is inhibited (TLR1, NME9, S100Z, black bars), one of the transcripts examined in a and b (MAPK1, grey bars), and a control transcript (BOP1, white bars). The results are presented as fold change with respect to wild-type Nck1 and are presented as mean ± standard deviation. The asterisk (*) indicates p
    Figure Legend Snippet: A functional role for Nck1 in cap homeostasis. Triplicate cultures of U2OS cells were transfected with plasmids expressing HA-tagged wild-type Nck1, Nck1 mutated in the CE-binding domain (M3), or the 5′-kinase-binding domain (M2). Western blots showing overexpression of each of these proteins are in Figure S4 . The appearance of uncapped forms of transcripts in the “capping inhibited” pool (grey bars) was determined by their recovery on streptavidin beads after ligation of an RNA adapter and hybridization to a biotinylated antisense DNA oligonucleotide [13] . Each preparation contained an equal amount of uncapped human β-globin mRNA as an internal control and RNA recovered from M3 (A) and M2 (B) expressing cells was analyzed by qRT-PCR for four transcripts that accumulate uncapped forms in cells that are inhibited for cytoplasmic capping (DNAJB1, ILF2, MAPK1, RAB1A). The results are normalized to the signal from cells expressing wild-type Nck1. RNA from M3 (C) or M2 (D) expressing cells was also analyzed by qRT-PCR for three transcripts of the “uninduced” pool whose steady state levels are reduced when cytoplasmic capping is inhibited (TLR1, NME9, S100Z, black bars), one of the transcripts examined in a and b (MAPK1, grey bars), and a control transcript (BOP1, white bars). The results are presented as fold change with respect to wild-type Nck1 and are presented as mean ± standard deviation. The asterisk (*) indicates p

    Techniques Used: Functional Assay, Transfection, Expressing, Binding Assay, Western Blot, Over Expression, Ligation, Hybridization, Quantitative RT-PCR, Standard Deviation

    Identification of the CE and 5′-kinase binding domains. (A) The organization of Nck1 (wild type [WT]) is shown together with a series of plasmids expressing HA-tagged forms with inactivating mutations (black box) in each of the functional domains. (B) HEK293 cells were co-transfected with plasmids expressing the indicated forms of Nck1 and bio-cCE. Protein recovered on streptavidin beads was analyzed by Western blotting with antibodies to the Myc tag on bio-cCE and the HA tag on Nck1. (C) Cells were co-transfected with plasmids expressing bio-cCE and HA-tagged wild type Nck1 (WT) or Nck1 with inactivating mutations in the third SH3 domain (M3) or all 3 SH3 domains (3SH3M). Protein recovered on streptavidin beads was analyzed by Western blotting with Alexafluor800-coupled streptavidin (cCE) and anti-HA (Nck1) antibody. Kinase activity was assayed by incubating the recovered proteins with a 23 nt 5′-monophosphate RNA and γ-[ 32 P]ATP, and capping activity was assayed by incubating recovered proteins with with ATP and α-[ 32 P]GTP. The products of each reaction were separated on a denaturing polyacrylamide/urea gel and visualized by autoradiography. (D) HEK293 cells were transfected with the plasmids expressing HA-tagged forms of wild-type Nck1, or Nck1 with inactivating mutations in the first (M1) and second (M2) SH3 domains. Complexes recovered on anti-HA beads were analyzed by Western blotting (upper panels), and for 5′-kinase activity as in (C).
    Figure Legend Snippet: Identification of the CE and 5′-kinase binding domains. (A) The organization of Nck1 (wild type [WT]) is shown together with a series of plasmids expressing HA-tagged forms with inactivating mutations (black box) in each of the functional domains. (B) HEK293 cells were co-transfected with plasmids expressing the indicated forms of Nck1 and bio-cCE. Protein recovered on streptavidin beads was analyzed by Western blotting with antibodies to the Myc tag on bio-cCE and the HA tag on Nck1. (C) Cells were co-transfected with plasmids expressing bio-cCE and HA-tagged wild type Nck1 (WT) or Nck1 with inactivating mutations in the third SH3 domain (M3) or all 3 SH3 domains (3SH3M). Protein recovered on streptavidin beads was analyzed by Western blotting with Alexafluor800-coupled streptavidin (cCE) and anti-HA (Nck1) antibody. Kinase activity was assayed by incubating the recovered proteins with a 23 nt 5′-monophosphate RNA and γ-[ 32 P]ATP, and capping activity was assayed by incubating recovered proteins with with ATP and α-[ 32 P]GTP. The products of each reaction were separated on a denaturing polyacrylamide/urea gel and visualized by autoradiography. (D) HEK293 cells were transfected with the plasmids expressing HA-tagged forms of wild-type Nck1, or Nck1 with inactivating mutations in the first (M1) and second (M2) SH3 domains. Complexes recovered on anti-HA beads were analyzed by Western blotting (upper panels), and for 5′-kinase activity as in (C).

    Techniques Used: Binding Assay, Expressing, Functional Assay, Transfection, Western Blot, Activity Assay, Autoradiography

    11) Product Images from "Targeting of Liposomes via PSGL1 for Enhanced Tumor Accumulation"

    Article Title: Targeting of Liposomes via PSGL1 for Enhanced Tumor Accumulation

    Journal: Pharmaceutical Research

    doi: 10.1007/s11095-012-0875-5

    Production of a protein G-streptavidin conjugate for linkage of immunoglobulin G Fc containing ligands to liposomes. ( a ) Outline of the strategy for the linkage of liposomes presenting PEG-biotin to the targeting ligand PSGL1-Fc via a linker comprised of streptavidin (for biotin binding) and protein G (for Fc binding). ( b ) Coomassie stained SDSPAGE of protein G-streptavidin conjugates formed at ratios of; lane 1 = 3 streptavidin : 1 protein G, lane 2 = 1 streptavidin : 4 protein G, lane 3 = free protein G in reaction buffers, lane 4 = free protein G direct from stock. ( c ) Probing of nitrocellulose with biotin-HRP; lane 1 = 3 streptavidin : 1 protein G, lane 2 = 1 streptavidin : 4 protein G, lane 3 = free protein G in reaction buffers. ( d ) Probing of nitrocellulose with Fc-HRP; lane 1 = 3 streptavidin : 1 protein G, lane 2 = 1 streptavidin : 4 protein G, lane 3 = free protein G in reaction buffers.
    Figure Legend Snippet: Production of a protein G-streptavidin conjugate for linkage of immunoglobulin G Fc containing ligands to liposomes. ( a ) Outline of the strategy for the linkage of liposomes presenting PEG-biotin to the targeting ligand PSGL1-Fc via a linker comprised of streptavidin (for biotin binding) and protein G (for Fc binding). ( b ) Coomassie stained SDSPAGE of protein G-streptavidin conjugates formed at ratios of; lane 1 = 3 streptavidin : 1 protein G, lane 2 = 1 streptavidin : 4 protein G, lane 3 = free protein G in reaction buffers, lane 4 = free protein G direct from stock. ( c ) Probing of nitrocellulose with biotin-HRP; lane 1 = 3 streptavidin : 1 protein G, lane 2 = 1 streptavidin : 4 protein G, lane 3 = free protein G in reaction buffers. ( d ) Probing of nitrocellulose with Fc-HRP; lane 1 = 3 streptavidin : 1 protein G, lane 2 = 1 streptavidin : 4 protein G, lane 3 = free protein G in reaction buffers.

    Techniques Used: Binding Assay, Staining

    12) Product Images from "Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex"

    Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081734

    Affinity purification of SBP-tagged AIMP1, AIMP2 and KARS. ( A ) Schematic diagram of AIMP1, AIMP2, and KARS constructs for affinity purification. S/FLAG/SBP tags were attached to the N-terminus of cloned genes. ( B ) Expression of AIMP1, AIMP2, and KARS tagged with S/FLAG/SBP in HEK 293T cells were confirmed by immunoblotting analysis using anti-FLAG antibody. Closed arrowheads (◀) indicate AIMP1, AIMP2 and KARS. ( C ) Streptavidin affinity purification was carried out and 10 % of the eluted samples from HEK 293T and HCT-8 cells were visualized by protein staining. One of three biological replicates is shown and the bait proteins are marked with red arrows. ( D ) 90 % of elution was separated on SDS-PAGE to about 1-cm distance and divided into three fractions each. Then, tryptic peptides were recovered from each gel bands and analyzed by LC-MS/MS. SAINT algorithm was used to calculate the likelihood of true interaction of identified proteins. M; Mock, A1; AIMP1, A2; AIMP2, K; KARS. ‘Mock’ is a vector having the S/FLAG/SBP tag only without target genes.
    Figure Legend Snippet: Affinity purification of SBP-tagged AIMP1, AIMP2 and KARS. ( A ) Schematic diagram of AIMP1, AIMP2, and KARS constructs for affinity purification. S/FLAG/SBP tags were attached to the N-terminus of cloned genes. ( B ) Expression of AIMP1, AIMP2, and KARS tagged with S/FLAG/SBP in HEK 293T cells were confirmed by immunoblotting analysis using anti-FLAG antibody. Closed arrowheads (◀) indicate AIMP1, AIMP2 and KARS. ( C ) Streptavidin affinity purification was carried out and 10 % of the eluted samples from HEK 293T and HCT-8 cells were visualized by protein staining. One of three biological replicates is shown and the bait proteins are marked with red arrows. ( D ) 90 % of elution was separated on SDS-PAGE to about 1-cm distance and divided into three fractions each. Then, tryptic peptides were recovered from each gel bands and analyzed by LC-MS/MS. SAINT algorithm was used to calculate the likelihood of true interaction of identified proteins. M; Mock, A1; AIMP1, A2; AIMP2, K; KARS. ‘Mock’ is a vector having the S/FLAG/SBP tag only without target genes.

    Techniques Used: Affinity Purification, Construct, Clone Assay, Expressing, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation

    TARSL2 as a member of ARS core complex. ( A , B ) TARSL2 and TARS were detected in AIMP1, AIMP2, and KARS immunoprecipitates of HEK 293T ( A ) and HCT-8 cells ( B ). EPRS was used as a positive control. Actin was used for loading control. TCL; total cell lysate, SA pull down; streptavidin pull down. ( C ) Endogenous EPRS was co-immunoprecipitated with TARSL2. Cell lysate (500 μg) was immunoprecipitated with the antibodies against TARSL2, TARS, and IgG and probed for EPRS, TARSL2 and TARS. ( D ) Reciprocal co-immunoprecipitation. Cell lysate (500 μg) was immunoprecipitated with the antibodies against EPRS, AIMP1, AIMP2, KARS and IgG and probed for TARSL2. IgG was used for immunoprecipitation control. M; Mock, E; ERPS, A1; AIMP1, A2; AIMP2, K; KARS, In; Input, TL2; TARSL2, T; TARS, IgG(R); rabbit IgG, IgG(M); mouse IgG. Closed arrowheads (▶) indicate TARSL2 and TARS.
    Figure Legend Snippet: TARSL2 as a member of ARS core complex. ( A , B ) TARSL2 and TARS were detected in AIMP1, AIMP2, and KARS immunoprecipitates of HEK 293T ( A ) and HCT-8 cells ( B ). EPRS was used as a positive control. Actin was used for loading control. TCL; total cell lysate, SA pull down; streptavidin pull down. ( C ) Endogenous EPRS was co-immunoprecipitated with TARSL2. Cell lysate (500 μg) was immunoprecipitated with the antibodies against TARSL2, TARS, and IgG and probed for EPRS, TARSL2 and TARS. ( D ) Reciprocal co-immunoprecipitation. Cell lysate (500 μg) was immunoprecipitated with the antibodies against EPRS, AIMP1, AIMP2, KARS and IgG and probed for TARSL2. IgG was used for immunoprecipitation control. M; Mock, E; ERPS, A1; AIMP1, A2; AIMP2, K; KARS, In; Input, TL2; TARSL2, T; TARS, IgG(R); rabbit IgG, IgG(M); mouse IgG. Closed arrowheads (▶) indicate TARSL2 and TARS.

    Techniques Used: Positive Control, Immunoprecipitation

    13) Product Images from "A Mixed Stimuli-Responsive Magnetic and Gold Nanoparticle System for Rapid Purification, Enrichment, and Detection of Biomarkers"

    Article Title: A Mixed Stimuli-Responsive Magnetic and Gold Nanoparticle System for Rapid Purification, Enrichment, and Detection of Biomarkers

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc100180q

    Magnetic enrichment of fluorescent-streptavidin by AuNP/mNP mixtures. AF-750 labeled-streptavidin fluorescence and AuNP absorbance were measured after processing variable volumes of 50% human plasma containing 5 nM fluorescent-streptavidin. In all cases,
    Figure Legend Snippet: Magnetic enrichment of fluorescent-streptavidin by AuNP/mNP mixtures. AF-750 labeled-streptavidin fluorescence and AuNP absorbance were measured after processing variable volumes of 50% human plasma containing 5 nM fluorescent-streptavidin. In all cases,

    Techniques Used: Labeling, Fluorescence

    Effect of increasing sample volume on LFIA signal. Increasing sample volumes from 100–500 μ L each with the same concentration of streptavidin (10 ng/mL) were processed using the AuNP/mNP bioseparaton method. 500 μ L of 50% human
    Figure Legend Snippet: Effect of increasing sample volume on LFIA signal. Increasing sample volumes from 100–500 μ L each with the same concentration of streptavidin (10 ng/mL) were processed using the AuNP/mNP bioseparaton method. 500 μ L of 50% human

    Techniques Used: Concentration Assay

    Lateral flow immunoassay standard curve. (Top) Image of the flow strip. (Bottom) Variable amounts of streptavidin spiked into 50% human plasma samples were captured at a fixed sample volume of 200 μ L. The background-subtracted mean pixel intensity
    Figure Legend Snippet: Lateral flow immunoassay standard curve. (Top) Image of the flow strip. (Bottom) Variable amounts of streptavidin spiked into 50% human plasma samples were captured at a fixed sample volume of 200 μ L. The background-subtracted mean pixel intensity

    Techniques Used: Flow Cytometry, Stripping Membranes

    Dual nanoparticle magnetic separation scheme. AuNPs coated with biotinylated diblock copolymers bind to streptavidin spiked into 50% human plasma. mNPs coated with homo-pNIPAAm are added, and the temperature is raised above the polymer LCST. Mixed streptavidin-AuNP/mNP
    Figure Legend Snippet: Dual nanoparticle magnetic separation scheme. AuNPs coated with biotinylated diblock copolymers bind to streptavidin spiked into 50% human plasma. mNPs coated with homo-pNIPAAm are added, and the temperature is raised above the polymer LCST. Mixed streptavidin-AuNP/mNP

    Techniques Used:

    14) Product Images from "Recognition of host Clr-b by the inhibitory NKR-P1B receptor provides a basis for missing-self recognition"

    Article Title: Recognition of host Clr-b by the inhibitory NKR-P1B receptor provides a basis for missing-self recognition

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06989-2

    Energetic basis of the NKR-P1B:Clr-b interaction. a BWZ cells (top) or HEK293T cells (bottom) were transduced or transfected, respectively, with empty vector (dashed line) or Clr-b-expressing vector (shaded gray), and stained using anti-Clr-b antibody (left) or NKR-P1B tetramers (right) and analyzed by flow cytometry. b HEK293T cells were transfected with vector expressing Clr-b (pIRES2-EGFP), and 48 h later were stained with NKR-P1B mutant tetramers. The GFP expression measures transfection efficiency and PE measures binding by PE-tetramers. Gates were set up using untransfected cells (HEK293T) and cells transfected with empty pIRES2-EGFP (Vector) that were stained with NKR-P1B tetramer (WT) or Streptavidin-PE (SA-PE). Labels on the top left correspond to point mutation on the NKR-P1B molecule, whereas italicized numbers correspond to mean fluorescence intensity of NKR-P1B. c Cells were transfected with constructs expressing NKR-P1B mutants, and 48 h later were used as stimulators to BWZ.CD3ζ/Clr-b reporters. Co-cultures were setup using a 1:1 stimulators: reporters ratio, and the next day were assayed for production of β-galactosidase using colorimetric assay. d , e NKR-P1B dimer mutants were transfected into HEK293T cells and used in BWZ assays using 3-fold dilutions of stimulators against BWZ.CD3ζ/Clr-b reporters (left) or BWZ.CD3ζ/m12 Smith reporters (right). Significant differences are shown between the WT and mutant allele for each graph. Data were analyzed using ( c ) one-way ANOVA [F(15,48) = 51.88, p
    Figure Legend Snippet: Energetic basis of the NKR-P1B:Clr-b interaction. a BWZ cells (top) or HEK293T cells (bottom) were transduced or transfected, respectively, with empty vector (dashed line) or Clr-b-expressing vector (shaded gray), and stained using anti-Clr-b antibody (left) or NKR-P1B tetramers (right) and analyzed by flow cytometry. b HEK293T cells were transfected with vector expressing Clr-b (pIRES2-EGFP), and 48 h later were stained with NKR-P1B mutant tetramers. The GFP expression measures transfection efficiency and PE measures binding by PE-tetramers. Gates were set up using untransfected cells (HEK293T) and cells transfected with empty pIRES2-EGFP (Vector) that were stained with NKR-P1B tetramer (WT) or Streptavidin-PE (SA-PE). Labels on the top left correspond to point mutation on the NKR-P1B molecule, whereas italicized numbers correspond to mean fluorescence intensity of NKR-P1B. c Cells were transfected with constructs expressing NKR-P1B mutants, and 48 h later were used as stimulators to BWZ.CD3ζ/Clr-b reporters. Co-cultures were setup using a 1:1 stimulators: reporters ratio, and the next day were assayed for production of β-galactosidase using colorimetric assay. d , e NKR-P1B dimer mutants were transfected into HEK293T cells and used in BWZ assays using 3-fold dilutions of stimulators against BWZ.CD3ζ/Clr-b reporters (left) or BWZ.CD3ζ/m12 Smith reporters (right). Significant differences are shown between the WT and mutant allele for each graph. Data were analyzed using ( c ) one-way ANOVA [F(15,48) = 51.88, p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Cytometry, Mutagenesis, Binding Assay, Fluorescence, Construct, Colorimetric Assay

    15) Product Images from "CD6 modulates thymocyte selection and peripheral T cell homeostasis"

    Article Title: CD6 modulates thymocyte selection and peripheral T cell homeostasis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151785

    Modulation of TCR-induced responses from CD6 −/− developing and mature T cells. (A, left) Fluorescence microscopy assessment of SOCE over time in Fura-2 AM–loaded thymocytes from CD6 +/+ ( n = 199 cells, open circles), CD6 −/− ( n = 188 cells, gray filled circles), and CD5 −/− ( n = 198 cells, black filled circles) mice after exposure to 145.2C11 mAb (αCD3) and Fc-specific antiserum (αIgG; n = 4 mice/genotype). (right) SOCE as represented by mean ± SEM of area under curve (AUC) from the same experiment. (B) FACS analysis of i[Ca 2+ ] over time in Indo-1 AM–loaded DP, CD8 + SP, and CD4 + SP cells from CD6 +/+ (black solid line) and CD6 −/− (gray solid line) mice exposed to biotinylated 145.2C11 mAb (αCD3) plus Streptavidin (SAv). Data are representative of three independent experiments. (C, left) FACS analysis of CD5 expression on DP thymocytes from CD6 +/+ (black solid line), CD5 +/− (black dashed line), and CD6 −/− (gray shaded line). (right) FACS analysis of i[Ca 2+ ] over time of Indo-1 AM–loaded DP cells from CD6 +/+ (black solid line), CD5 +/− (black dashed line), CD6 −/− (gray solid line) and CD5 −/− (gray dashed line) mice, upon exposure to αCD3 mAb plus SAv, as assessed in B. (D) Dot density graphs showing the percentage (mean ± SEM) of major thymocyte subsets (left) and of LN CD4 + and CD8 + T EM and T CM subsets (right) from CD5 +/− (black filled circles), CD6 +/+ (open circles), and CD6 −/− (gray filled circles) mice. The results shown are from one representative of two experiments performed. **, P
    Figure Legend Snippet: Modulation of TCR-induced responses from CD6 −/− developing and mature T cells. (A, left) Fluorescence microscopy assessment of SOCE over time in Fura-2 AM–loaded thymocytes from CD6 +/+ ( n = 199 cells, open circles), CD6 −/− ( n = 188 cells, gray filled circles), and CD5 −/− ( n = 198 cells, black filled circles) mice after exposure to 145.2C11 mAb (αCD3) and Fc-specific antiserum (αIgG; n = 4 mice/genotype). (right) SOCE as represented by mean ± SEM of area under curve (AUC) from the same experiment. (B) FACS analysis of i[Ca 2+ ] over time in Indo-1 AM–loaded DP, CD8 + SP, and CD4 + SP cells from CD6 +/+ (black solid line) and CD6 −/− (gray solid line) mice exposed to biotinylated 145.2C11 mAb (αCD3) plus Streptavidin (SAv). Data are representative of three independent experiments. (C, left) FACS analysis of CD5 expression on DP thymocytes from CD6 +/+ (black solid line), CD5 +/− (black dashed line), and CD6 −/− (gray shaded line). (right) FACS analysis of i[Ca 2+ ] over time of Indo-1 AM–loaded DP cells from CD6 +/+ (black solid line), CD5 +/− (black dashed line), CD6 −/− (gray solid line) and CD5 −/− (gray dashed line) mice, upon exposure to αCD3 mAb plus SAv, as assessed in B. (D) Dot density graphs showing the percentage (mean ± SEM) of major thymocyte subsets (left) and of LN CD4 + and CD8 + T EM and T CM subsets (right) from CD5 +/− (black filled circles), CD6 +/+ (open circles), and CD6 −/− (gray filled circles) mice. The results shown are from one representative of two experiments performed. **, P

    Techniques Used: Fluorescence, Microscopy, Mouse Assay, FACS, Expressing

    16) Product Images from "An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides"

    Article Title: An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1186/s40425-018-0339-5

    Test for antigenicity of svL4 and sv6D. Anti-sera were generated in rabbits against KLH-conjugates of the sequences of svL4 or sv6D and diluted 1:10. Mouse sera were collected after alternate-day injections of svL4 over 3 months, diluted 1:1 with PBS and added to protein-A/G-coated wells. Biotinylated svL4 or sv6D was added to wells and bound peptide was detected with a streptavidin-peroxidase conjugate. The figure includes values ± S.D. for sera from 8 treated mice in each group assayed separately
    Figure Legend Snippet: Test for antigenicity of svL4 and sv6D. Anti-sera were generated in rabbits against KLH-conjugates of the sequences of svL4 or sv6D and diluted 1:10. Mouse sera were collected after alternate-day injections of svL4 over 3 months, diluted 1:1 with PBS and added to protein-A/G-coated wells. Biotinylated svL4 or sv6D was added to wells and bound peptide was detected with a streptavidin-peroxidase conjugate. The figure includes values ± S.D. for sera from 8 treated mice in each group assayed separately

    Techniques Used: Generated, Mouse Assay

    sv6D as a mimetic of GalNAc. a Inhibition by sv6D of binding of multivalent GalNAc-PAA (GlycoTech Corp., Frederick, MD) to recombinant rat CLEC10A and human ASGPR-1. The reaction mixture included approximately 200 pmoles of biotinylated GalNc-PAA and increasing concentrations of peptide. The figure includes values ± S.D. from 3 independent experiments. b Biotinylated sv6D or GalNAc-PAA were incubated with rabbit antiserum raised against the 6D sequence (NQHTPR) conjugated to KLH. Binding was detected with streptavidin-peroxidase conjugate. Similar data were obtained in 2 experiments. c Inhibition by EGTA of binding of sv6D (red, yellow) or svL4 (dark green, light green) to CLEC10A and ASGPR-1, respectively. EGTA (1 mM) was added to the final concentrations indicated to assays. Retention of bound CLEC10A was determined by incubation with biotinylated anti-CLEC10A (goat IgG, R D Systems) and detection with streptavidin-peroxidase conjugate (blue). Similar data were obtained in 3 experiments. d Binding of svL4 and sv6D to human recombinant CLEC10A and ASGPR-1 as a function of concentration of peptide in the assay. The figure is representative of 4 separate assays. K D values ± S.D. from reciprocal plots of these data are provided in the text
    Figure Legend Snippet: sv6D as a mimetic of GalNAc. a Inhibition by sv6D of binding of multivalent GalNAc-PAA (GlycoTech Corp., Frederick, MD) to recombinant rat CLEC10A and human ASGPR-1. The reaction mixture included approximately 200 pmoles of biotinylated GalNc-PAA and increasing concentrations of peptide. The figure includes values ± S.D. from 3 independent experiments. b Biotinylated sv6D or GalNAc-PAA were incubated with rabbit antiserum raised against the 6D sequence (NQHTPR) conjugated to KLH. Binding was detected with streptavidin-peroxidase conjugate. Similar data were obtained in 2 experiments. c Inhibition by EGTA of binding of sv6D (red, yellow) or svL4 (dark green, light green) to CLEC10A and ASGPR-1, respectively. EGTA (1 mM) was added to the final concentrations indicated to assays. Retention of bound CLEC10A was determined by incubation with biotinylated anti-CLEC10A (goat IgG, R D Systems) and detection with streptavidin-peroxidase conjugate (blue). Similar data were obtained in 3 experiments. d Binding of svL4 and sv6D to human recombinant CLEC10A and ASGPR-1 as a function of concentration of peptide in the assay. The figure is representative of 4 separate assays. K D values ± S.D. from reciprocal plots of these data are provided in the text

    Techniques Used: Inhibition, Binding Assay, Recombinant, Incubation, Sequencing, Concentration Assay

    17) Product Images from "Diffractometric Detection of Proteins using Microbead-based Rolling Circle Amplification"

    Article Title: Diffractometric Detection of Proteins using Microbead-based Rolling Circle Amplification

    Journal: Analytical chemistry

    doi: 10.1021/ac901716d

    (a) Schematic of microbead-based aptamer sandwich assay without RCA. Biotinylated aptamer was immobilized on streptavidin functionalized periodic patterns. PDGF-BB was introduced, and sandwiched by another biotinylated aptamer. Streptavidin conjugated
    Figure Legend Snippet: (a) Schematic of microbead-based aptamer sandwich assay without RCA. Biotinylated aptamer was immobilized on streptavidin functionalized periodic patterns. PDGF-BB was introduced, and sandwiched by another biotinylated aptamer. Streptavidin conjugated

    Techniques Used:

    (a)-(e) Optical micrographs of the self-assembled diffraction gratings formed by streptavidin-labeled beads with varying PDGF-BB concentration (10 pM – 100 nM). (f) Grating with no PDGF-BB.
    Figure Legend Snippet: (a)-(e) Optical micrographs of the self-assembled diffraction gratings formed by streptavidin-labeled beads with varying PDGF-BB concentration (10 pM – 100 nM). (f) Grating with no PDGF-BB.

    Techniques Used: Labeling, Concentration Assay

    (a) Schematic of RCA-based microbead detection assay in combination with aptamers. A biotinylated anti-PDGF-B specific aptamer is immobilized on streptavidin coated periodic patterns. PDGF-BB is introduced and captured by the aptamer. An aptamer-primer
    Figure Legend Snippet: (a) Schematic of RCA-based microbead detection assay in combination with aptamers. A biotinylated anti-PDGF-B specific aptamer is immobilized on streptavidin coated periodic patterns. PDGF-BB is introduced and captured by the aptamer. An aptamer-primer

    Techniques Used: Detection Assay

    18) Product Images from "Site-specific mapping and quantification of protein S-sulfenylation in cells"

    Article Title: Site-specific mapping and quantification of protein S-sulfenylation in cells

    Journal: Nature communications

    doi: 10.1038/ncomms5776

    Site-specific mapping of protein S -sulfenylation in cells ( a ) Workflow for selective labeling and analysis of protein S -sulfenylation in living cells. S -sulfenylated cysteines in intact RKO cells were labeled with the dimedone-based probe, DYn-2. Cell proteins then were digested with trypsin and labeled peptides were conjugated with azide biotin reagent, captured with streptavidin beads, and released by photocleavage of the biotin linker. The released DYn-2-triazohexanoic acid-modified peptides were analyzed by LC-MS/MS. ( b ) Characteristic fragmentation of modified peptides (upper, left) and a representative MS1 spectrum (upper, right) and HCD MS/MS spectrum (lower) of a DYn-2-triazohexanoic acid-modified peptide from S-sulfenylated PRDX6. The highlighted cysteine in the peptide sequence represents the S-sulfenylated site (C47) of PRDX6. A zoom window displays the high mass accuracy of three diagnostic fragment ion (DFI) peaks.
    Figure Legend Snippet: Site-specific mapping of protein S -sulfenylation in cells ( a ) Workflow for selective labeling and analysis of protein S -sulfenylation in living cells. S -sulfenylated cysteines in intact RKO cells were labeled with the dimedone-based probe, DYn-2. Cell proteins then were digested with trypsin and labeled peptides were conjugated with azide biotin reagent, captured with streptavidin beads, and released by photocleavage of the biotin linker. The released DYn-2-triazohexanoic acid-modified peptides were analyzed by LC-MS/MS. ( b ) Characteristic fragmentation of modified peptides (upper, left) and a representative MS1 spectrum (upper, right) and HCD MS/MS spectrum (lower) of a DYn-2-triazohexanoic acid-modified peptide from S-sulfenylated PRDX6. The highlighted cysteine in the peptide sequence represents the S-sulfenylated site (C47) of PRDX6. A zoom window displays the high mass accuracy of three diagnostic fragment ion (DFI) peaks.

    Techniques Used: Labeling, Modification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Diagnostic Assay

    19) Product Images from "Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules"

    Article Title: Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules

    Journal: Scientific Reports

    doi: 10.1038/srep10584

    Separation and mobility of protein molecules in the 3D nanowire structures. ( a ) Separation of (1) trypsin inhibitor (20.1 kDa), (2) protein A (45 kDa), (3) streptavidin (52.8 kDa), (4) β-galactosidase (116 kDa) and (5) fibrinogen (340 kDa). The electropherograms were obtained at 2000 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 500 V/cm. ( b ) The electropherograms of each type of protein molecule to verify the migration time of each separation peak ( E = 2000 V/cm, L = 500 μm). ( c ) Semi-log plot of electrophoretic mobility as a function of molecular weight under the applied electric field of 500V/cm.
    Figure Legend Snippet: Separation and mobility of protein molecules in the 3D nanowire structures. ( a ) Separation of (1) trypsin inhibitor (20.1 kDa), (2) protein A (45 kDa), (3) streptavidin (52.8 kDa), (4) β-galactosidase (116 kDa) and (5) fibrinogen (340 kDa). The electropherograms were obtained at 2000 μm from the entrance of the 3D nanowire structures. The applied electric field in the separation channel was 500 V/cm. ( b ) The electropherograms of each type of protein molecule to verify the migration time of each separation peak ( E = 2000 V/cm, L = 500 μm). ( c ) Semi-log plot of electrophoretic mobility as a function of molecular weight under the applied electric field of 500V/cm.

    Techniques Used: Migration, Molecular Weight

    20) Product Images from "Conditional ligands for Asian HLA variants facilitate the definition of CD8+ T-cell responses in acute and chronic viral diseases"

    Article Title: Conditional ligands for Asian HLA variants facilitate the definition of CD8+ T-cell responses in acute and chronic viral diseases

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201243088

    Peptide binding to Asian HLA variants. (A) Seven 15-mer peptides capable of stimulating T-cells reactive toward SARS-CoV, HBV, or DENV antigens were discovered by ELISPOT screening. All 18 possible 9-, 10-, and 11-mer truncated peptides imbedded in the 15-mer epitope that either tested positive in the HLA-stability assay (gray bars) or gave HLA tetramer staining (black bars) are indicated below the sequence. Numbering of the peptide fragments is provided in Supporting Information Fig. 3A . (B) Soluble HLA-A*02:01 molecules were ligand exchanged with truncated peptides from SARS NP44 15-mer peptide. The resulting complexes were captured on streptavidin-coated plates and probed for β2m as a marker for HLA complex stability. As controls, the photocleavable HLA molecules were treated with (+) or without (–) UV irradiation in the absence of rescue peptide (black bars). Peptides are labeled as capable (red bars) or incapable (white bars) of stabilizing the corresponding HLA. (C) Stabilization of soluble HLA-A*02:07 by truncations of the SARS NP44 15-mer peptide was assessed as in (B). (D) Stabilization of soluble HLA-B*40:01 by truncations of the SARS NP44 15-mer peptide was assessed as in (B). (E) Stabilization of soluble HLA-B*46:01 by truncations of the SARS NP44 15-mer peptide was assessed as in (B). (B–E) Data are shown as mean + SEM of four replicates.
    Figure Legend Snippet: Peptide binding to Asian HLA variants. (A) Seven 15-mer peptides capable of stimulating T-cells reactive toward SARS-CoV, HBV, or DENV antigens were discovered by ELISPOT screening. All 18 possible 9-, 10-, and 11-mer truncated peptides imbedded in the 15-mer epitope that either tested positive in the HLA-stability assay (gray bars) or gave HLA tetramer staining (black bars) are indicated below the sequence. Numbering of the peptide fragments is provided in Supporting Information Fig. 3A . (B) Soluble HLA-A*02:01 molecules were ligand exchanged with truncated peptides from SARS NP44 15-mer peptide. The resulting complexes were captured on streptavidin-coated plates and probed for β2m as a marker for HLA complex stability. As controls, the photocleavable HLA molecules were treated with (+) or without (–) UV irradiation in the absence of rescue peptide (black bars). Peptides are labeled as capable (red bars) or incapable (white bars) of stabilizing the corresponding HLA. (C) Stabilization of soluble HLA-A*02:07 by truncations of the SARS NP44 15-mer peptide was assessed as in (B). (D) Stabilization of soluble HLA-B*40:01 by truncations of the SARS NP44 15-mer peptide was assessed as in (B). (E) Stabilization of soluble HLA-B*46:01 by truncations of the SARS NP44 15-mer peptide was assessed as in (B). (B–E) Data are shown as mean + SEM of four replicates.

    Techniques Used: Binding Assay, Enzyme-linked Immunospot, Stability Assay, Staining, Sequencing, Marker, Irradiation, Labeling

    21) Product Images from "Biomolecular Surface Engineering of Pancreatic Islets with Thrombomodulin"

    Article Title: Biomolecular Surface Engineering of Pancreatic Islets with Thrombomodulin

    Journal: Acta biomaterialia

    doi: 10.1016/j.actbio.2010.01.027

    Immobilization of rTM on the islet surface via streptavidin-biotin interactions increases rates of activated protein C (APC) generation Upon combination biotinylation and subsequent incubation with streptavidin (Biotin + SA) islets were incubated with rTM-biotin at 3.5 µM for 1 hour, resulting in an approximately three-fold increase in the rate of APC generation relative to untreated controls (*p
    Figure Legend Snippet: Immobilization of rTM on the islet surface via streptavidin-biotin interactions increases rates of activated protein C (APC) generation Upon combination biotinylation and subsequent incubation with streptavidin (Biotin + SA) islets were incubated with rTM-biotin at 3.5 µM for 1 hour, resulting in an approximately three-fold increase in the rate of APC generation relative to untreated controls (*p

    Techniques Used: Incubation

    Site-specific biotinylation of recombinant human thrombomodulin (A) Upon reaction between rTM-N 3 and triphenylphosphine-PEG 3.4kD -biotin, SDS PAGE reveals the presence of two species separated by approximately 4 kD (Lane 1), corresponding to the desired biotinylated conjugate (*) and unreacted rTM-N 3 . A molecular weight shift was not observed in a parallel control reaction using rTM engineered without an azido group (Lane 2), demonstrating the specificity of the Staudinger ligation. (B) Western blot against human TM after initial conjugation (Lane 1) and subsequent purification (Lane 2). After purification via centrifugal dialysis and monomeric avidin chromatography, a single species corresponding to the expected molecular weight of the desired biotin-PEG-TM conjugate is observed (*). (C) Western blot against biotin using HRP-labeled streptavidin confirms biotinylation of the construct (*; Lane 1).
    Figure Legend Snippet: Site-specific biotinylation of recombinant human thrombomodulin (A) Upon reaction between rTM-N 3 and triphenylphosphine-PEG 3.4kD -biotin, SDS PAGE reveals the presence of two species separated by approximately 4 kD (Lane 1), corresponding to the desired biotinylated conjugate (*) and unreacted rTM-N 3 . A molecular weight shift was not observed in a parallel control reaction using rTM engineered without an azido group (Lane 2), demonstrating the specificity of the Staudinger ligation. (B) Western blot against human TM after initial conjugation (Lane 1) and subsequent purification (Lane 2). After purification via centrifugal dialysis and monomeric avidin chromatography, a single species corresponding to the expected molecular weight of the desired biotin-PEG-TM conjugate is observed (*). (C) Western blot against biotin using HRP-labeled streptavidin confirms biotinylation of the construct (*; Lane 1).

    Techniques Used: Recombinant, SDS Page, Molecular Weight, Ligation, Western Blot, Conjugation Assay, Purification, Avidin-Biotin Assay, Chromatography, Labeling, Construct

    22) Product Images from "Phospholipase Cγ1 is required for pre-TCR signal transduction and pre-T cell development"

    Article Title: Phospholipase Cγ1 is required for pre-TCR signal transduction and pre-T cell development

    Journal: European journal of immunology

    doi: 10.1002/eji.201646522

    PLCγ1 deficiency impaired pre-TCR mediated Ca 2+ flux and Erk activation. (A) Ca 2+ flux following pre-TCR stimulation is impaired in PLCγ1-deficient pre-T cells. The analysis was performed on gated YFP + TCRβ + DN3 (upper panel) and DN4 (lower panel) thymocytes. The arrows indicated the time when streptavidin was added to crosslink the biotin-anti-CD3 or when ionomycin was added. Data is a representative six independent experiments. (B) Erk phosphorylation following pre-TCR stimulation is impaired in PLCγ1-deficient pre-T cells. pErk analysis was gated on YFP + TCRβ + DN4 thymocytes. The upper panel represents the isotype control staining of anti-CD3 + anti-CD28 stimulated LckCre/YFP/PLCγ1 f/+ (solid line) and LckCre/YFP/PLCγ1 f/− (dashed line) YFP + TCRβ + DN4 thymocytes. The lower panel represents anti-pErk staining of the LckCre/YFP/PLCγ1 f/+ (solid line) and LckCre/YFP/PLCγ1 f/− (dashed line) YFP + TCRβ + DN4 thymocytes treated with medium (grey line) or stimulated with anti-CD3 + anti-CD28 (black line). Data is a representative of 3 independent experiments. (C) Statistics analysis of the ratio of pErk mean fluorescent intensity (MFI) between LckCre/YFP/PLCγ1 f/− (n=3) and LckCre/YFP/PLCγ1 f/+ thymocytes (n=3) when cells are treated with medium or stimulated with anti-CD3 + anti-CD28. Data are shown as mean +SD and are representative of three independent experiments. P =0.03 in Student’s t -test.
    Figure Legend Snippet: PLCγ1 deficiency impaired pre-TCR mediated Ca 2+ flux and Erk activation. (A) Ca 2+ flux following pre-TCR stimulation is impaired in PLCγ1-deficient pre-T cells. The analysis was performed on gated YFP + TCRβ + DN3 (upper panel) and DN4 (lower panel) thymocytes. The arrows indicated the time when streptavidin was added to crosslink the biotin-anti-CD3 or when ionomycin was added. Data is a representative six independent experiments. (B) Erk phosphorylation following pre-TCR stimulation is impaired in PLCγ1-deficient pre-T cells. pErk analysis was gated on YFP + TCRβ + DN4 thymocytes. The upper panel represents the isotype control staining of anti-CD3 + anti-CD28 stimulated LckCre/YFP/PLCγ1 f/+ (solid line) and LckCre/YFP/PLCγ1 f/− (dashed line) YFP + TCRβ + DN4 thymocytes. The lower panel represents anti-pErk staining of the LckCre/YFP/PLCγ1 f/+ (solid line) and LckCre/YFP/PLCγ1 f/− (dashed line) YFP + TCRβ + DN4 thymocytes treated with medium (grey line) or stimulated with anti-CD3 + anti-CD28 (black line). Data is a representative of 3 independent experiments. (C) Statistics analysis of the ratio of pErk mean fluorescent intensity (MFI) between LckCre/YFP/PLCγ1 f/− (n=3) and LckCre/YFP/PLCγ1 f/+ thymocytes (n=3) when cells are treated with medium or stimulated with anti-CD3 + anti-CD28. Data are shown as mean +SD and are representative of three independent experiments. P =0.03 in Student’s t -test.

    Techniques Used: Activation Assay, Staining

    23) Product Images from "Phosphorylation Modulates Aspartyl-(Asparaginyl)-β Hydroxylase Protein Expression, Catalytic Activity and Migration in Human Immature Neuronal Cerebellar Cells"

    Article Title: Phosphorylation Modulates Aspartyl-(Asparaginyl)-β Hydroxylase Protein Expression, Catalytic Activity and Migration in Human Immature Neuronal Cerebellar Cells

    Journal: Cell biology : research & therapy

    doi: 10.4172/2324-9293.1000133

    Effects of WT N-Myc-ASPH Over-expression on ASPH and Notch Pathway Protein Immunoreactivity: PNET2 cells transfected with recombinant plasmids carrying full-length cDNAs encoding (A, D, G, J) WT-ASPH, (B, E, H, K) GFP, or (C, F, I, L) Humbug (truncated form of ASPH) were harvested after 48 hours to generate cytospin preparations for immunofluorescence assays. Formalin-fixed, permeabilized cells were incubated with (A-C) A85G6-ASPH, (D-F) FB50-ASPH/Humbug, (G-I) Notch-1, or (J-L) Jagged-1 monoclonal antibodies. Immunoreactivity was detected with biotinylated secondary antibody and Streptavidin-conjugated Dylight 547 (green) or Dylight 647 (red). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Panels depict merged confocal microscopy images. (600× original magnification, 2× digitalzoom).
    Figure Legend Snippet: Effects of WT N-Myc-ASPH Over-expression on ASPH and Notch Pathway Protein Immunoreactivity: PNET2 cells transfected with recombinant plasmids carrying full-length cDNAs encoding (A, D, G, J) WT-ASPH, (B, E, H, K) GFP, or (C, F, I, L) Humbug (truncated form of ASPH) were harvested after 48 hours to generate cytospin preparations for immunofluorescence assays. Formalin-fixed, permeabilized cells were incubated with (A-C) A85G6-ASPH, (D-F) FB50-ASPH/Humbug, (G-I) Notch-1, or (J-L) Jagged-1 monoclonal antibodies. Immunoreactivity was detected with biotinylated secondary antibody and Streptavidin-conjugated Dylight 547 (green) or Dylight 647 (red). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Panels depict merged confocal microscopy images. (600× original magnification, 2× digitalzoom).

    Techniques Used: Over Expression, Transfection, Recombinant, Immunofluorescence, Incubation, Confocal Microscopy

    Effects of S/T→A mutations on ASPH protein expression and subcellular localization: PNET2 cells were transiently transfected with wildtype (WT) or a pointmutated (M#:S/T→A) N-Myc-ASPH cDNA. Myc-empty vector (EV) served as a negative control. The M19-H675Q mutant, disrupting ASPH’s catalytic activity, served as a positive control. Representative results obtained by immunofluorescence staining and confocal imaging of cells transfected with (A) WT, (B) M7-S24A, (C) M18-T748A, (D) M19-H675Q, or (E) EV and stained by immunofluorescence with anti-Myc. Immunoreactivity was detected with biotinylated secondary antibody and Streptavidin-conjugated Dylight 547 (red). Cells were counterstained with DAPI (blue). (Merged images: 600× magnification, 2× digital zoom).
    Figure Legend Snippet: Effects of S/T→A mutations on ASPH protein expression and subcellular localization: PNET2 cells were transiently transfected with wildtype (WT) or a pointmutated (M#:S/T→A) N-Myc-ASPH cDNA. Myc-empty vector (EV) served as a negative control. The M19-H675Q mutant, disrupting ASPH’s catalytic activity, served as a positive control. Representative results obtained by immunofluorescence staining and confocal imaging of cells transfected with (A) WT, (B) M7-S24A, (C) M18-T748A, (D) M19-H675Q, or (E) EV and stained by immunofluorescence with anti-Myc. Immunoreactivity was detected with biotinylated secondary antibody and Streptavidin-conjugated Dylight 547 (red). Cells were counterstained with DAPI (blue). (Merged images: 600× magnification, 2× digital zoom).

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Negative Control, Mutagenesis, Activity Assay, Positive Control, Immunofluorescence, Staining, Imaging

    24) Product Images from "TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering"

    Article Title: TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.09.051

    TSPAN5 Promotes NLG1 Clustering (A) Confocal images of dendrites from DIV 12 rat hippocampal cultured neurons transfected at DIV 5 with AP-NLG1, BirA-ER, and scrambled, Sh-TSPAN5, rescue, DLQ, or NIYF all co-expressing mCherry. Surface-applied Streptavidin-488 clusters shown in green. Scale bar, 5 μm. (B) Quantification of the cluster density (clusters/square micrometer) and average size (square micrometers) of Streptavidin-488 clusters. Scrambled, n = 19 neurons; Sh-TSPAN5, n = 20 neurons; rescue, n = 18 neurons; DLQ, n = 27 neurons; NIYF, n = 27 neurons. (C) Single-molecule tracking experiment with mStrav-647. Images of DIV 12 rat hippocampal cultured neurons transfected at DIV 5 with AP-NLG1, BirA-ER, and scrambled, Sh-TSPAN5, rescue, or PLM all co-expressing GFP. Top panels: widefield GFP signal of dendrites. Middle panels (intensity): super-resolved mStrav-647 detection maps as an intensity scale. Bottom panels (tracks): mStrav-647 trajectories shown in pseudocolors. Scale bar, 2 μm. (D) Quantification of single-molecule tracking. Left panel: quantification of global diffusion coefficient (square micrometers per second). Right panel: logarithmic distribution plot of diffusion coefficients of scrambled- (black), Sh-TSPAN5- (blue), rescue- (purple), or PLM-transfected neurons (green). Scrambled, n = 16 neurons; Sh-TSPAN5, n = 17 neurons; rescue, n = 16 neurons; PLM, n = 12 neurons. (E) Confocal images of DIV 14 cultured rat hippocampal neurons transfected at DIV 5 with HA-tagged NLG1 and either GFP-expressing scrambled or Sh-TSPAN5 constructs. Neurons treated from DIV 12 to 14 with either non-clustered neurexin1β-Fc (left panels) or neurexin1β-Fc pre-clustered with an unlabeled α-Fc antibody (right panels). Surface-applied α-HA antibody shown in red; surface-applied Alexa 647-labeled α-Fc shown in blue. Purple arrowheads show mushroom spines positive for clusters. (F) Pie charts of the proportion of stubby, thin, or mushroom spines relative to (E). Exact values are shown in Table S1 . See also Figures S4–S7 . Graphs represent mean ± SEM. In (F), ∗ p
    Figure Legend Snippet: TSPAN5 Promotes NLG1 Clustering (A) Confocal images of dendrites from DIV 12 rat hippocampal cultured neurons transfected at DIV 5 with AP-NLG1, BirA-ER, and scrambled, Sh-TSPAN5, rescue, DLQ, or NIYF all co-expressing mCherry. Surface-applied Streptavidin-488 clusters shown in green. Scale bar, 5 μm. (B) Quantification of the cluster density (clusters/square micrometer) and average size (square micrometers) of Streptavidin-488 clusters. Scrambled, n = 19 neurons; Sh-TSPAN5, n = 20 neurons; rescue, n = 18 neurons; DLQ, n = 27 neurons; NIYF, n = 27 neurons. (C) Single-molecule tracking experiment with mStrav-647. Images of DIV 12 rat hippocampal cultured neurons transfected at DIV 5 with AP-NLG1, BirA-ER, and scrambled, Sh-TSPAN5, rescue, or PLM all co-expressing GFP. Top panels: widefield GFP signal of dendrites. Middle panels (intensity): super-resolved mStrav-647 detection maps as an intensity scale. Bottom panels (tracks): mStrav-647 trajectories shown in pseudocolors. Scale bar, 2 μm. (D) Quantification of single-molecule tracking. Left panel: quantification of global diffusion coefficient (square micrometers per second). Right panel: logarithmic distribution plot of diffusion coefficients of scrambled- (black), Sh-TSPAN5- (blue), rescue- (purple), or PLM-transfected neurons (green). Scrambled, n = 16 neurons; Sh-TSPAN5, n = 17 neurons; rescue, n = 16 neurons; PLM, n = 12 neurons. (E) Confocal images of DIV 14 cultured rat hippocampal neurons transfected at DIV 5 with HA-tagged NLG1 and either GFP-expressing scrambled or Sh-TSPAN5 constructs. Neurons treated from DIV 12 to 14 with either non-clustered neurexin1β-Fc (left panels) or neurexin1β-Fc pre-clustered with an unlabeled α-Fc antibody (right panels). Surface-applied α-HA antibody shown in red; surface-applied Alexa 647-labeled α-Fc shown in blue. Purple arrowheads show mushroom spines positive for clusters. (F) Pie charts of the proportion of stubby, thin, or mushroom spines relative to (E). Exact values are shown in Table S1 . See also Figures S4–S7 . Graphs represent mean ± SEM. In (F), ∗ p

    Techniques Used: Cell Culture, Transfection, Expressing, Diffusion-based Assay, Construct, Labeling

    25) Product Images from "Sequence-specific inhibition of Dicer measured with a force-based microarray for RNA ligands"

    Article Title: Sequence-specific inhibition of Dicer measured with a force-based microarray for RNA ligands

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1455

    Schematics of the Molecular Force Assay. ( A ) The molecular complex is built up by covalently attaching the lowest strand to a glass slide and, subsequently, binding the pre-hybridized upper duplex to the lowest strand. The fluorophor Cy5 is conjugated to a poly-T sequence connecting the two duplexes. The upper strand is labelled with Cy3 so that a FRET signal provides a measure for a correctly hybridized molecular construct. The ‘RNA up’ geometry is defined with the DNA complex attached to the glass slide and the RNA duplex constituting the upper part. A biotin–streptavidin–biotin bond links the molecular complex to the upper surface, a soft PDMS stamp. Upon retracting the PDMS stamp, a force builds up in the molecular constructs and unzips the duplexes until the weaker of the two bonds in series ruptures. Note that in this format Cy5 serves as marker for those molecular complexes which remain intact. ( B ) In the setup, the contact device is mounted on an inverted microscope. The PDMS stamp features a micropattern that facilitates leveling and drainage of liquid during the contact and separation process. The oligonucleotide constructs are spotted in a 4 × 4 pattern, and fluorescence intensities are measured before and after the contact and separation process. After separation the fluorescence intensities of the molecules remaining on the glass and the PDMS surface add up to the total fluorescence intensity measured at the beginning. ( C ) Nucleic acid sequences of the molecular constructs in both configurations.
    Figure Legend Snippet: Schematics of the Molecular Force Assay. ( A ) The molecular complex is built up by covalently attaching the lowest strand to a glass slide and, subsequently, binding the pre-hybridized upper duplex to the lowest strand. The fluorophor Cy5 is conjugated to a poly-T sequence connecting the two duplexes. The upper strand is labelled with Cy3 so that a FRET signal provides a measure for a correctly hybridized molecular construct. The ‘RNA up’ geometry is defined with the DNA complex attached to the glass slide and the RNA duplex constituting the upper part. A biotin–streptavidin–biotin bond links the molecular complex to the upper surface, a soft PDMS stamp. Upon retracting the PDMS stamp, a force builds up in the molecular constructs and unzips the duplexes until the weaker of the two bonds in series ruptures. Note that in this format Cy5 serves as marker for those molecular complexes which remain intact. ( B ) In the setup, the contact device is mounted on an inverted microscope. The PDMS stamp features a micropattern that facilitates leveling and drainage of liquid during the contact and separation process. The oligonucleotide constructs are spotted in a 4 × 4 pattern, and fluorescence intensities are measured before and after the contact and separation process. After separation the fluorescence intensities of the molecules remaining on the glass and the PDMS surface add up to the total fluorescence intensity measured at the beginning. ( C ) Nucleic acid sequences of the molecular constructs in both configurations.

    Techniques Used: Binding Assay, Sequencing, Construct, Marker, Inverted Microscopy, Fluorescence

    26) Product Images from "A structural model for K2P potassium channels based on 23 pairs of interacting sites and continuum electrostatics"

    Article Title: A structural model for K2P potassium channels based on 23 pairs of interacting sites and continuum electrostatics

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200910235

    Lysine in P1 or P2 disrupts ΔK 2P Ø channel function but not surface expression. Wild-type (WT), S104K, or T216K ΔK 2P Ø channels were studied by expression in Xenopus oocytes. (A) Sample recordings by two-electrode voltage clamp. The bath solution contained 100 mM KCl (see Materials and methods). Topology insets: blue circles denote S104K in P1 or T216K in P2. Protocol inset: holding voltage of −77 mV with 250-ms steps of 15 mV from −135 to 60 mV followed by a 100-ms step to −135 mV every 2 s. (B) Western blot analyses of channels bearing C-terminal 1D4 tags detected with anti-1D4 antibodies. Channel protein was detected among total soluble protein (left) or surface proteins isolated by biotinylation and purification with streptavidin beads (right) after SDS-PAGE (see Materials and methods). Control samples were obtained from naive oocytes. Note that monomer subunits show an anomalous apparent mass of 62 (predicted mass of 37 kD) and 56 kD after deglycosylation with peptide– N -glycosidase F (not depicted); two linked subunits migrate at 97 and 89 kD after peptide– N -glycosidase F (not depicted).
    Figure Legend Snippet: Lysine in P1 or P2 disrupts ΔK 2P Ø channel function but not surface expression. Wild-type (WT), S104K, or T216K ΔK 2P Ø channels were studied by expression in Xenopus oocytes. (A) Sample recordings by two-electrode voltage clamp. The bath solution contained 100 mM KCl (see Materials and methods). Topology insets: blue circles denote S104K in P1 or T216K in P2. Protocol inset: holding voltage of −77 mV with 250-ms steps of 15 mV from −135 to 60 mV followed by a 100-ms step to −135 mV every 2 s. (B) Western blot analyses of channels bearing C-terminal 1D4 tags detected with anti-1D4 antibodies. Channel protein was detected among total soluble protein (left) or surface proteins isolated by biotinylation and purification with streptavidin beads (right) after SDS-PAGE (see Materials and methods). Control samples were obtained from naive oocytes. Note that monomer subunits show an anomalous apparent mass of 62 (predicted mass of 37 kD) and 56 kD after deglycosylation with peptide– N -glycosidase F (not depicted); two linked subunits migrate at 97 and 89 kD after peptide– N -glycosidase F (not depicted).

    Techniques Used: Expressing, Mass Spectrometry, Western Blot, Isolation, Purification, SDS Page

    27) Product Images from "Nanoplasmonic mid-infrared biosensor for in vitro protein secondary structure detection"

    Article Title: Nanoplasmonic mid-infrared biosensor for in vitro protein secondary structure detection

    Journal: Light, Science & Applications

    doi: 10.1038/lsa.2017.29

    Secondary structure components of the amide I absorption band of streptavidin immobilized via biotin binding. The three minima correspond to native β-sheets (within 1615–1645 cm −1 and 1680–1695 cm −1 ) and β turns (within 1665–1690 cm −1 ) of secondary structures of the immobilized protein measured in phosphate buffer saline (PBS), on one array with L : 1900 nm and P : 2.5 μm.
    Figure Legend Snippet: Secondary structure components of the amide I absorption band of streptavidin immobilized via biotin binding. The three minima correspond to native β-sheets (within 1615–1645 cm −1 and 1680–1695 cm −1 ) and β turns (within 1665–1690 cm −1 ) of secondary structures of the immobilized protein measured in phosphate buffer saline (PBS), on one array with L : 1900 nm and P : 2.5 μm.

    Techniques Used: Binding Assay

    28) Product Images from "Microsphere-based interferometric optical probe"

    Article Title: Microsphere-based interferometric optical probe

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07029-9

    Microenvironmental sensing. a Schematic illustration for the binding of streptavidin on a biotin-functionalized reflectophore. Upon binding, diameter increases by ~10 nm. b Fluorescence images before and after loading fluorescent streptavidin. Note that fluorescence was localized specifically at the surface of the reflectophore. c SpeRe measurements on control and streptavidin-bound reflectophores. The arrows indicate the spectral peaks. Data are represented as mean (open circle) and the standard deviation (shaded region) was acquired from 30 repeated SpeRe measurements. The solid lines are the best-fit simulated spectra ( R 2 in mean ± standard deviation: 0.98 ± 0.005 for “control”, 0.98 ± 0.005 for “+SA”). d Experimental setup for sensing electric field with a liquid-crystal reflectophore. Nematic liquid-crystal droplet is immersed in PDMS and sandwiched between conductive ITO glasses coupled to a function generator. e Liquid-crystal reflectophore imaged by bright-field, polarization, and reflectance microscopy. Scale bar, 4 μm. f Spectral reflectance of liquid-crystal reflectophore in response to applied electric field. Color bar represents normalized reflectance
    Figure Legend Snippet: Microenvironmental sensing. a Schematic illustration for the binding of streptavidin on a biotin-functionalized reflectophore. Upon binding, diameter increases by ~10 nm. b Fluorescence images before and after loading fluorescent streptavidin. Note that fluorescence was localized specifically at the surface of the reflectophore. c SpeRe measurements on control and streptavidin-bound reflectophores. The arrows indicate the spectral peaks. Data are represented as mean (open circle) and the standard deviation (shaded region) was acquired from 30 repeated SpeRe measurements. The solid lines are the best-fit simulated spectra ( R 2 in mean ± standard deviation: 0.98 ± 0.005 for “control”, 0.98 ± 0.005 for “+SA”). d Experimental setup for sensing electric field with a liquid-crystal reflectophore. Nematic liquid-crystal droplet is immersed in PDMS and sandwiched between conductive ITO glasses coupled to a function generator. e Liquid-crystal reflectophore imaged by bright-field, polarization, and reflectance microscopy. Scale bar, 4 μm. f Spectral reflectance of liquid-crystal reflectophore in response to applied electric field. Color bar represents normalized reflectance

    Techniques Used: Binding Assay, Fluorescence, Standard Deviation, Microscopy

    29) Product Images from "A Low-Cost, High-Performance System for Fluorescence Lateral Flow Assays"

    Article Title: A Low-Cost, High-Performance System for Fluorescence Lateral Flow Assays

    Journal: Biosensors

    doi: 10.3390/bios3040360

    Alexa Fluor 532 has a good ratio of signal to nonspecific binding (S/NSB) compared to Atto 430LS. Each fluorophore is conjugated to streptavidin, spotted on nitrocellulose and the signal read in the breadboard (signal, blue diamonds). Strips of nitrocellulose that have been blocked with BSA are immersed in each solution and read in the breadboard (nonspecific binding, magenta squares). The ratios of the two slopes are reported as the S/NSB ratio.
    Figure Legend Snippet: Alexa Fluor 532 has a good ratio of signal to nonspecific binding (S/NSB) compared to Atto 430LS. Each fluorophore is conjugated to streptavidin, spotted on nitrocellulose and the signal read in the breadboard (signal, blue diamonds). Strips of nitrocellulose that have been blocked with BSA are immersed in each solution and read in the breadboard (nonspecific binding, magenta squares). The ratios of the two slopes are reported as the S/NSB ratio.

    Techniques Used: Binding Assay

    Images and plot of absorbance lateral flow analysis of hCG. Lateral flow strips were spotted with goat anti-hCG, then dipped successively in a dilution series of hCG, followed by gold streptavidin mixed with biotinylated mouse anti-hCG, followed by buffer. Each concentration of hCG was tested in triplicate.
    Figure Legend Snippet: Images and plot of absorbance lateral flow analysis of hCG. Lateral flow strips were spotted with goat anti-hCG, then dipped successively in a dilution series of hCG, followed by gold streptavidin mixed with biotinylated mouse anti-hCG, followed by buffer. Each concentration of hCG was tested in triplicate.

    Techniques Used: Flow Cytometry, Concentration Assay

    Images and data for fluorescence lateral flow analysis of hCG. Lateral flow strips were spotted with goat anti-hCG, then dipped successively in a dilution series of hCG, followed by RPE streptavidin mixed with biotinylated mouse anti-hCG, followed by buffer. Each concentration of hCG was tested in triplicate.
    Figure Legend Snippet: Images and data for fluorescence lateral flow analysis of hCG. Lateral flow strips were spotted with goat anti-hCG, then dipped successively in a dilution series of hCG, followed by RPE streptavidin mixed with biotinylated mouse anti-hCG, followed by buffer. Each concentration of hCG was tested in triplicate.

    Techniques Used: Fluorescence, Flow Cytometry, Concentration Assay

    Fluorescence lateral flow images and plot. A spot rather than the conventional stripe of streptavidin was applied to the nitrocellulose. The spot diameter was approximately 3 mm. Dilutions of biotinylated BSA, followed by R-PE streptavidin, followed by buffer were absorbed onto the strips. Each concentration was tested in triplicate. Images were obtained in a breadboard equipped with an iPhone 4 and ProCamera app; a sample of the images is shown on the right. Image analysis was done with Image J and the results plotted.
    Figure Legend Snippet: Fluorescence lateral flow images and plot. A spot rather than the conventional stripe of streptavidin was applied to the nitrocellulose. The spot diameter was approximately 3 mm. Dilutions of biotinylated BSA, followed by R-PE streptavidin, followed by buffer were absorbed onto the strips. Each concentration was tested in triplicate. Images were obtained in a breadboard equipped with an iPhone 4 and ProCamera app; a sample of the images is shown on the right. Image analysis was done with Image J and the results plotted.

    Techniques Used: Fluorescence, Flow Cytometry, Concentration Assay

    Photobleaching of R-PE streptavidin (pink) and Alexa Fluor 532 streptavidin (blue). The compounds were spotted on nitrocellulose and exposed to constant illumination with a 505 nm LED. Images were collected at time intervals. ( a ) Data was normalized to the initial values; ( b ) A plot of the natural logarithm of the signal. Half-lives were determined by the equation t 1/2 = ln2/k, where k is the negative slope of the natural log plot.
    Figure Legend Snippet: Photobleaching of R-PE streptavidin (pink) and Alexa Fluor 532 streptavidin (blue). The compounds were spotted on nitrocellulose and exposed to constant illumination with a 505 nm LED. Images were collected at time intervals. ( a ) Data was normalized to the initial values; ( b ) A plot of the natural logarithm of the signal. Half-lives were determined by the equation t 1/2 = ln2/k, where k is the negative slope of the natural log plot.

    Techniques Used:

    Absorbance lateral flow images and plot. The strips were spotted with streptavidin. Dilutions of biotinylated BSA, followed by gold-labeled streptavidin, followed by buffer were absorbed on the strips. Each concentration was tested in triplicate. Images were obtained with the camera of an iPhone 4. Image analysis was done with Image J and the results plotted. A sample of the images is shown on the right.
    Figure Legend Snippet: Absorbance lateral flow images and plot. The strips were spotted with streptavidin. Dilutions of biotinylated BSA, followed by gold-labeled streptavidin, followed by buffer were absorbed on the strips. Each concentration was tested in triplicate. Images were obtained with the camera of an iPhone 4. Image analysis was done with Image J and the results plotted. A sample of the images is shown on the right.

    Techniques Used: Flow Cytometry, Labeling, Concentration Assay

    30) Product Images from "Translocation of the Na+/H+ exchanger 1 (NHE1) in cardiomyocyte responses to insulin and energy-status signalling"

    Article Title: Translocation of the Na+/H+ exchanger 1 (NHE1) in cardiomyocyte responses to insulin and energy-status signalling

    Journal: Biochemical Journal

    doi: 10.1042/BJ20100717

    Effects of hypoxia, oligomycin treatment and contraction on the abundance of NHE1 levels at the cell surface ( A ) Isolated cardiomyocytes were incubated in hypoxic buffer for 15 min, or treated with 5 μmol/l oligomycin for 60 min or were electrically stimulated to contract for 5 min. When indicated, cells were pre-incubated with 100 nmol/l wortmannin for 15 min. When the combined effect of insulin stimulation and hypoxia was studied, cardiomyocytes were first incubated with 30 nmol/l insulin for 30 min and insulin was then maintained throughout the hypoxic treatment. When the combined effect of insulin and oligomycin treatment was examined, after 30 min of oligomycin treatment 30 nmol/l insulin was added for the remaining 30 min of the oligomycin treatment. In each experiment, control cells were left untreated or incubated with 30 nmol/l insulin for 30 min. After the treatments, cardiomyocytes were cooled to 15 °C, labelled without (no biotin control, NBC) or with 180 μmol/l Sulfo-NHS-SS-Biotin. Membrane samples were solubilized (20 μg was taken for loading control, bottom panel), biotinylated protein was precipitated with immobilized streptavidin, the precipitated proteins were separated by SDS/PAGE and NHE1 was detected by Western blot analysis (top panel). The blots are representative of three to five independent experiments. ( B ) Quantification data from Western blot results. The histogram represents the mean + S.E.M from three to five independent experiments. * P
    Figure Legend Snippet: Effects of hypoxia, oligomycin treatment and contraction on the abundance of NHE1 levels at the cell surface ( A ) Isolated cardiomyocytes were incubated in hypoxic buffer for 15 min, or treated with 5 μmol/l oligomycin for 60 min or were electrically stimulated to contract for 5 min. When indicated, cells were pre-incubated with 100 nmol/l wortmannin for 15 min. When the combined effect of insulin stimulation and hypoxia was studied, cardiomyocytes were first incubated with 30 nmol/l insulin for 30 min and insulin was then maintained throughout the hypoxic treatment. When the combined effect of insulin and oligomycin treatment was examined, after 30 min of oligomycin treatment 30 nmol/l insulin was added for the remaining 30 min of the oligomycin treatment. In each experiment, control cells were left untreated or incubated with 30 nmol/l insulin for 30 min. After the treatments, cardiomyocytes were cooled to 15 °C, labelled without (no biotin control, NBC) or with 180 μmol/l Sulfo-NHS-SS-Biotin. Membrane samples were solubilized (20 μg was taken for loading control, bottom panel), biotinylated protein was precipitated with immobilized streptavidin, the precipitated proteins were separated by SDS/PAGE and NHE1 was detected by Western blot analysis (top panel). The blots are representative of three to five independent experiments. ( B ) Quantification data from Western blot results. The histogram represents the mean + S.E.M from three to five independent experiments. * P

    Techniques Used: Isolation, Incubation, SDS Page, Western Blot

    Immunocytochemical analysis of NHE1 subcellular distribution in insulin-, hypoxia- and contraction-stimulated cardiomyocytes Isolated cardiomyocytes were maintained in a basal unstimulated state or incubated in one of the following conditions: with 30 nmol/l insulin for 30 min, in a hypoxic buffer for 15 min, with 5 μmol/l oligomycin for 60 min, or electrically stimulated to contract for 5 min. After the treatments, cells were washed briefly and fixed with 4% (w/v) paraformaldehyde. After permeabilization with 0.1% saponin, cells were incubated with rabbit anti-rat NHE1 antibody and Alexa Fluor® 488-conjugated anti-rabbit IgG (green) (left-hand panels) and visualized by confocal microscopy. All cells were viewed in approximately the same focal plane. For quantification of the changes in the NHE1 levels at the sarcolemma, non-permeabilized cells were labelled with LcH and streptavidin–Alexa Fluor® 633 conjugate (red) prior to proceeding to the immunolabelling of NHE1 (middle panels). Merged images are shown in the right-hand panels. Control cells were incubated with rabbit anti-rat NHE1 antibody that had been pre-incubated with the purified NHE1 fragment that was used as the antigen. Immunofluorescent images shown are representative from 8–31 cells examined per condition from three to five independent experiments. Scale bars=20 μm.
    Figure Legend Snippet: Immunocytochemical analysis of NHE1 subcellular distribution in insulin-, hypoxia- and contraction-stimulated cardiomyocytes Isolated cardiomyocytes were maintained in a basal unstimulated state or incubated in one of the following conditions: with 30 nmol/l insulin for 30 min, in a hypoxic buffer for 15 min, with 5 μmol/l oligomycin for 60 min, or electrically stimulated to contract for 5 min. After the treatments, cells were washed briefly and fixed with 4% (w/v) paraformaldehyde. After permeabilization with 0.1% saponin, cells were incubated with rabbit anti-rat NHE1 antibody and Alexa Fluor® 488-conjugated anti-rabbit IgG (green) (left-hand panels) and visualized by confocal microscopy. All cells were viewed in approximately the same focal plane. For quantification of the changes in the NHE1 levels at the sarcolemma, non-permeabilized cells were labelled with LcH and streptavidin–Alexa Fluor® 633 conjugate (red) prior to proceeding to the immunolabelling of NHE1 (middle panels). Merged images are shown in the right-hand panels. Control cells were incubated with rabbit anti-rat NHE1 antibody that had been pre-incubated with the purified NHE1 fragment that was used as the antigen. Immunofluorescent images shown are representative from 8–31 cells examined per condition from three to five independent experiments. Scale bars=20 μm.

    Techniques Used: Isolation, Incubation, Confocal Microscopy, Purification

    Treatment with insulin increases the abundance of NHE1 at the cell surface through a PI3K-dependent mechanism ( A ) Isolated cardiomyocytes were incubated for 30 min with or without 30 nmol/l insulin. Where indicated, cells were pre-treated for 15 min with 100 nmol/l wortmannin before the addition of insulin. Cardiomyocytes were then cooled to 15 °C, labelled with 180 μmol/l Sulfo-NHS-SS-Biotin, washed and membranes prepared. Membranes were solubilized (20 μg of protein was taken for a loading control), biotinylated protein was precipitated with immobilized streptavidin, the precipitated proteins were separated by SDS/PAGE and NHE1 was detected by Western blot analysis. The blots are representative of five independent experiments. ( B ) Quantification data from the Western blot results. The histogram represents the means ± S.E.M. from five independent experiments. * P
    Figure Legend Snippet: Treatment with insulin increases the abundance of NHE1 at the cell surface through a PI3K-dependent mechanism ( A ) Isolated cardiomyocytes were incubated for 30 min with or without 30 nmol/l insulin. Where indicated, cells were pre-treated for 15 min with 100 nmol/l wortmannin before the addition of insulin. Cardiomyocytes were then cooled to 15 °C, labelled with 180 μmol/l Sulfo-NHS-SS-Biotin, washed and membranes prepared. Membranes were solubilized (20 μg of protein was taken for a loading control), biotinylated protein was precipitated with immobilized streptavidin, the precipitated proteins were separated by SDS/PAGE and NHE1 was detected by Western blot analysis. The blots are representative of five independent experiments. ( B ) Quantification data from the Western blot results. The histogram represents the means ± S.E.M. from five independent experiments. * P

    Techniques Used: Isolation, Incubation, SDS Page, Western Blot

    31) Product Images from "In situ transduction of target cells on solid surfaces by immobilized viral vectors"

    Article Title: In situ transduction of target cells on solid surfaces by immobilized viral vectors

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-3-4

    Immobilization efficiency of biotinylated adenoviral vectors on streptavidin-coated wells. (A) Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors (1 × 10 6 – 5 × 10 6 viral particles in 50 μl PBST per well) were incubated in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for 2 h at 25°C for immobilization. The solution of each well, which contained unbound viral particles, was collected and analyzed for the infectivity on D-17 cells (○). The same numbers of biotinylated Ad5.CMV-LacZ without application to streptavidin-coated wells were also analyzed for the infectivity on D-17 cells (●). The infectivity of immobilized viral particles was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied to each well. The percentage of immobilized viral particles in the total is shown at the top. Each data point shown is the average + SD (n = 3). (B) Biotinylated Ad5.CMV-LacZ, prepared as above, was incubated in streptavidin-coated wells (2.5 × 10 6 viral particles per well) for 30 min at 25°C. The solution of each well was collected, and the wells were washed three times with PBST. Then, the same number of fresh biotinylated Ad5.CMV-LacZ was incubated in the same manner as above. These steps were repeated several more times. The solution in the well, which contained unbound viral particles, was collected after each addition cycle and titrated on D-17 cells (open bars). The Control bar shows the total infectivity of biotinylated Ad5.CMV-LacZ that was applied to wells at each addition cycle. The infectivity of immobilized viral particles (solid bars) was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied at each addition cycle. The percentage of immobilized viral particles in the total at each addition cycle is shown at the top. Each data point shown is the average ± SD (n = 3).
    Figure Legend Snippet: Immobilization efficiency of biotinylated adenoviral vectors on streptavidin-coated wells. (A) Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors (1 × 10 6 – 5 × 10 6 viral particles in 50 μl PBST per well) were incubated in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for 2 h at 25°C for immobilization. The solution of each well, which contained unbound viral particles, was collected and analyzed for the infectivity on D-17 cells (○). The same numbers of biotinylated Ad5.CMV-LacZ without application to streptavidin-coated wells were also analyzed for the infectivity on D-17 cells (●). The infectivity of immobilized viral particles was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied to each well. The percentage of immobilized viral particles in the total is shown at the top. Each data point shown is the average + SD (n = 3). (B) Biotinylated Ad5.CMV-LacZ, prepared as above, was incubated in streptavidin-coated wells (2.5 × 10 6 viral particles per well) for 30 min at 25°C. The solution of each well was collected, and the wells were washed three times with PBST. Then, the same number of fresh biotinylated Ad5.CMV-LacZ was incubated in the same manner as above. These steps were repeated several more times. The solution in the well, which contained unbound viral particles, was collected after each addition cycle and titrated on D-17 cells (open bars). The Control bar shows the total infectivity of biotinylated Ad5.CMV-LacZ that was applied to wells at each addition cycle. The infectivity of immobilized viral particles (solid bars) was calculated by subtraction of the infectivity of unbound viral particles from the total infectivity of Ad5.CMV-LacZ applied at each addition cycle. The percentage of immobilized viral particles in the total at each addition cycle is shown at the top. Each data point shown is the average ± SD (n = 3).

    Techniques Used: Incubation, Infection

    In situ transduction of target cells by adenoviral vectors immobilized on a solid surface. Ad5.CMV-LacZ was treated with varying concentrations of sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylated reagent. The resulting viral vectors (5 × 10 6 viral particles per well) were incubated in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for 2 h at 25°C for immobilization. After unbound viral particles were removed, D-17 cells (8 × 10 3 cells per well) were placed in the wells and cultured at 37°C for 48 h. Cells were fixed with glutaraldehyde and stained for the expression of the lac Z gene (●). Biotinylated Ad5.CMV-LacZ (5 × 10 6 viral particles per well) was used free in solution as a control (○). Data shown are representative of six independent experiments.
    Figure Legend Snippet: In situ transduction of target cells by adenoviral vectors immobilized on a solid surface. Ad5.CMV-LacZ was treated with varying concentrations of sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylated reagent. The resulting viral vectors (5 × 10 6 viral particles per well) were incubated in streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for 2 h at 25°C for immobilization. After unbound viral particles were removed, D-17 cells (8 × 10 3 cells per well) were placed in the wells and cultured at 37°C for 48 h. Cells were fixed with glutaraldehyde and stained for the expression of the lac Z gene (●). Biotinylated Ad5.CMV-LacZ (5 × 10 6 viral particles per well) was used free in solution as a control (○). Data shown are representative of six independent experiments.

    Techniques Used: In Situ, Transduction, Incubation, Cell Culture, Staining, Expressing

    Effect of the amount of adenoviral vectors on in situ transduction of target cells on a solid surface. Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors (2.5 × 10 5 – 2.5 × 10 8 viral particles per well) were added to streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for immobilization, followed by the removal of unbound viral particles. Then, D-17 cells (8 × 10 2 cells per well) were placed on the wells and cultured at 37°C for 48 h. Cells were fixed with glutaraldehyde and stained for the expression of the lac Z gene (●). Unmodified Ad5.CMV-LacZ was used free in solution as a control (○). Data shown are representative of five independent experiments.
    Figure Legend Snippet: Effect of the amount of adenoviral vectors on in situ transduction of target cells on a solid surface. Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors (2.5 × 10 5 – 2.5 × 10 8 viral particles per well) were added to streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for immobilization, followed by the removal of unbound viral particles. Then, D-17 cells (8 × 10 2 cells per well) were placed on the wells and cultured at 37°C for 48 h. Cells were fixed with glutaraldehyde and stained for the expression of the lac Z gene (●). Unmodified Ad5.CMV-LacZ was used free in solution as a control (○). Data shown are representative of five independent experiments.

    Techniques Used: In Situ, Transduction, Cell Culture, Staining, Expressing

    Effect of the natural permissivity of target cells for adenoviral infection on in situ transduction of the cells by adenoviral vectors immobilized on a solid surface. A, D-17 cells (canine osteosarcoma cell line that is highly permissive to adenoviral infection); B, C6 cells (rat glioma cell line that is poorly permissive to adenoviral infection). Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors were added to streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for immobilization, followed by the removal of unbound viral particles. Target cells (8 × 10 3 cells per well) were placed on the wells and cultured at 37°C for 48 h. Cells were fixed with glutaraldehyde and stained for the expression of the lac Z gene (●). Unmodified Ad5.CMV-LacZ was used free in solution as a control (○). Data shown in A and B are representative of three and four, respectively, independent experiments.
    Figure Legend Snippet: Effect of the natural permissivity of target cells for adenoviral infection on in situ transduction of the cells by adenoviral vectors immobilized on a solid surface. A, D-17 cells (canine osteosarcoma cell line that is highly permissive to adenoviral infection); B, C6 cells (rat glioma cell line that is poorly permissive to adenoviral infection). Ad5.CMV-LacZ was treated with 15 μg/ml sulfo-NHS-LC-biotin, followed by the removal of non-virion-associated biotinylation reagent. Varying numbers of the resulting biotinylated adenoviral vectors were added to streptavidin-coated wells (well diameter, 0.64 cm; Reacti-Bind Streptavidin Coated Polystyrene Wells) for immobilization, followed by the removal of unbound viral particles. Target cells (8 × 10 3 cells per well) were placed on the wells and cultured at 37°C for 48 h. Cells were fixed with glutaraldehyde and stained for the expression of the lac Z gene (●). Unmodified Ad5.CMV-LacZ was used free in solution as a control (○). Data shown in A and B are representative of three and four, respectively, independent experiments.

    Techniques Used: Infection, In Situ, Transduction, Cell Culture, Staining, Expressing

    32) Product Images from "Vaccinia virus virulence factor N1 can be ubiquitylated on multiple lysine residues"

    Article Title: Vaccinia virus virulence factor N1 can be ubiquitylated on multiple lysine residues

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.065664-0

    Generation of a recombinant TAP-tagged N1 virus. (a) Fusion of a TAP [streptavidin (STREP) and FLAG] tag at the C terminus of N1. (b) Infection of BS-C-1 cells with VACV strain WR, a recombinant VACV lacking the N1L gene (vΔN1) or a recombinant VACV expressing TAP-tagged N1 (vN1.TAP) at 2 p.f.u. per cell for 16 h. Whole-cell lysates were resolved by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. Molecular mass markers are also included.
    Figure Legend Snippet: Generation of a recombinant TAP-tagged N1 virus. (a) Fusion of a TAP [streptavidin (STREP) and FLAG] tag at the C terminus of N1. (b) Infection of BS-C-1 cells with VACV strain WR, a recombinant VACV lacking the N1L gene (vΔN1) or a recombinant VACV expressing TAP-tagged N1 (vN1.TAP) at 2 p.f.u. per cell for 16 h. Whole-cell lysates were resolved by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. Molecular mass markers are also included.

    Techniques Used: Recombinant, FLAG-tag, Infection, Expressing, SDS Page

    33) Product Images from "HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats"

    Article Title: HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0130811

    HD5 mAb is specific to B27 2 , binds to recombinant B27 2 , and blocks interaction with immunoregulatory cell receptors. (A) ELISA results showed the specificity of HD5 and HD6 to B27 2 homodimers when challenged for different recombinant HLA class I complexes (-A1,-B7,-B13,-C7,-B27, and-B27 2 ). Control HC10 and W6/32 antibodies were used as positive and negative control, respectively. (B) Direct ELISA against HLA-G and B27 2 homodimers using HD5, HD6, HC10 and W6/32 antibodies showed specificity of HD5 and HD6 to immobilized B27 2 homodimers but not to HLA-G homodimers. (C-E) Recombinant B27 2 , B27-free-heavy chain and HLA-B27 heterotrimers were immobilized into chips for kinetic characterization by SPR. C) HD5 and ligand (B27 2 ) have a K d of 0.32 nM. Immobilized free-heavy chains (D) or HLA–B27 heterotrimers (E) did not interact with HD5 and K d values were not fitted. (F) Blocking competition experiments in SPR were performed by immobilizing B27 2 to a streptavidin chip followed by injection of HD5 to form B27 2 -HD5 complexes. Injections of LILRB2, KIR3DL2 and Pirb were assessed and binding events recorded.
    Figure Legend Snippet: HD5 mAb is specific to B27 2 , binds to recombinant B27 2 , and blocks interaction with immunoregulatory cell receptors. (A) ELISA results showed the specificity of HD5 and HD6 to B27 2 homodimers when challenged for different recombinant HLA class I complexes (-A1,-B7,-B13,-C7,-B27, and-B27 2 ). Control HC10 and W6/32 antibodies were used as positive and negative control, respectively. (B) Direct ELISA against HLA-G and B27 2 homodimers using HD5, HD6, HC10 and W6/32 antibodies showed specificity of HD5 and HD6 to immobilized B27 2 homodimers but not to HLA-G homodimers. (C-E) Recombinant B27 2 , B27-free-heavy chain and HLA-B27 heterotrimers were immobilized into chips for kinetic characterization by SPR. C) HD5 and ligand (B27 2 ) have a K d of 0.32 nM. Immobilized free-heavy chains (D) or HLA–B27 heterotrimers (E) did not interact with HD5 and K d values were not fitted. (F) Blocking competition experiments in SPR were performed by immobilizing B27 2 to a streptavidin chip followed by injection of HD5 to form B27 2 -HD5 complexes. Injections of LILRB2, KIR3DL2 and Pirb were assessed and binding events recorded.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control, Direct ELISA, SPR Assay, Blocking Assay, Chromatin Immunoprecipitation, Injection, Binding Assay

    Pro-inflammatory cytokines increase cell-surface B27 2 homodimers. (A-E) Cell cultures of leukocyte populations were analyzed by flow cytometry for presence of cell-surface B27 2 homodimers after stimulation with cytokines. HD6-biotinylated and streptavidin APC were used for detection. Mean fluorescence intensity (MFI) values were plotted from data analysis. Statistical analysis was plotted against “without cytokines” data values. Values are expressed as mean±SEM. *p
    Figure Legend Snippet: Pro-inflammatory cytokines increase cell-surface B27 2 homodimers. (A-E) Cell cultures of leukocyte populations were analyzed by flow cytometry for presence of cell-surface B27 2 homodimers after stimulation with cytokines. HD6-biotinylated and streptavidin APC were used for detection. Mean fluorescence intensity (MFI) values were plotted from data analysis. Statistical analysis was plotted against “without cytokines” data values. Values are expressed as mean±SEM. *p

    Techniques Used: Flow Cytometry, Cytometry, Fluorescence

    Detection of cell-surface B27 2 in leukocyte populations of HLA-B27 transgenic rats at different ages. (A) Representative flow cytometry analysis of cell-surface B27 2 expression in leukocytes populations of Tg rats at different ages (6, 15, 23 and 30 weeks) from spleen and MLNs. WT leukocytes represent the control population where B27 2 is absent. (B) MFI values plotted of positive B27 2 stains from splenocytes. (C) MFI values plotted of positive B27 2 stains from MLNs. Detection of cell-surface B27 2 homodimers was performed using HD6-biotinylated and detected by streptavidin-APC. HD6 had been previously assessed as an antibody capable of recognizing cell-surface B27 2 in human [ 12 ] and rat [ 41 ] leukocyte populations. Antibody panels: CD4+ T-cells (+CD3, +CD4), CD8+ T-cells (+CD3, +CD8), NK (+CD161a,—lineage), B cells (+CD45RA,-lineage) and Monocytes (+CD172a,—RP-1).
    Figure Legend Snippet: Detection of cell-surface B27 2 in leukocyte populations of HLA-B27 transgenic rats at different ages. (A) Representative flow cytometry analysis of cell-surface B27 2 expression in leukocytes populations of Tg rats at different ages (6, 15, 23 and 30 weeks) from spleen and MLNs. WT leukocytes represent the control population where B27 2 is absent. (B) MFI values plotted of positive B27 2 stains from splenocytes. (C) MFI values plotted of positive B27 2 stains from MLNs. Detection of cell-surface B27 2 homodimers was performed using HD6-biotinylated and detected by streptavidin-APC. HD6 had been previously assessed as an antibody capable of recognizing cell-surface B27 2 in human [ 12 ] and rat [ 41 ] leukocyte populations. Antibody panels: CD4+ T-cells (+CD3, +CD4), CD8+ T-cells (+CD3, +CD8), NK (+CD161a,—lineage), B cells (+CD45RA,-lineage) and Monocytes (+CD172a,—RP-1).

    Techniques Used: Transgenic Assay, Flow Cytometry, Cytometry, Expressing

    Reduced accumulation of cell-surface B27 2 molecules in Tg-HD5 rats. Analysis of cell-surface B27 2 from spleen and MLN was performed using HD6-biotinylated and detected by streptavidin-APC in WT-littermates and HLA-B27 rat groups at 15 and 23 weeks . (A-B) MFI values plotted of positive B27 2 stains from flow cytometry analyzed splenocytes (n = 5) (A) and MLN (n = 5) (B) subpopulations. Values are expressed as mean±SEM. *p
    Figure Legend Snippet: Reduced accumulation of cell-surface B27 2 molecules in Tg-HD5 rats. Analysis of cell-surface B27 2 from spleen and MLN was performed using HD6-biotinylated and detected by streptavidin-APC in WT-littermates and HLA-B27 rat groups at 15 and 23 weeks . (A-B) MFI values plotted of positive B27 2 stains from flow cytometry analyzed splenocytes (n = 5) (A) and MLN (n = 5) (B) subpopulations. Values are expressed as mean±SEM. *p

    Techniques Used: Flow Cytometry, Cytometry

    34) Product Images from "Design and characterization of a protein superagonist of IL-15 fused with IL-15R? and a high-affinity T cell receptor"

    Article Title: Design and characterization of a protein superagonist of IL-15 fused with IL-15R? and a high-affinity T cell receptor

    Journal: Biotechnology progress

    doi: 10.1002/btpr.1631

    Cytokine-receptor binding of scTv:cytokine fusion proteins ) (A) CTLL-2 or (B) MC57 were incubated with the indicated concentration of scTv:cytokine fusion, then washed and stained with SIY/K b streptavidin tetramer.
    Figure Legend Snippet: Cytokine-receptor binding of scTv:cytokine fusion proteins ) (A) CTLL-2 or (B) MC57 were incubated with the indicated concentration of scTv:cytokine fusion, then washed and stained with SIY/K b streptavidin tetramer.

    Techniques Used: Binding Assay, Incubation, Concentration Assay, Staining

    Peptide/MHC-specific binding of scTv:cytokine fusion proteins (A–C) T2-K b cells were loaded with either null (OVA) or specific (SIY) peptide and then incubated with the indicated scTv:cytokine fusions. After washing, cells were incubated with antibodies to the fused cytokines: (A) biotinylated anti-IL-15 antibody, followed by streptavidin-Alexa 488, (B) biotinylated rat anti-IL-2 antibody, followed by with streptavidin-PE and (C) polyclonal goat anti-IL-15Ra antibody, detected with biotinylated polyclonal rabbit anti-goat antibody followed by streptavidin-Alexa 647. In each panel, the solid line trace represents staining by the primary antibody and secondary reagent and the filled, gray histogram represents staining by the secondary reagent only.
    Figure Legend Snippet: Peptide/MHC-specific binding of scTv:cytokine fusion proteins (A–C) T2-K b cells were loaded with either null (OVA) or specific (SIY) peptide and then incubated with the indicated scTv:cytokine fusions. After washing, cells were incubated with antibodies to the fused cytokines: (A) biotinylated anti-IL-15 antibody, followed by streptavidin-Alexa 488, (B) biotinylated rat anti-IL-2 antibody, followed by with streptavidin-PE and (C) polyclonal goat anti-IL-15Ra antibody, detected with biotinylated polyclonal rabbit anti-goat antibody followed by streptavidin-Alexa 647. In each panel, the solid line trace represents staining by the primary antibody and secondary reagent and the filled, gray histogram represents staining by the secondary reagent only.

    Techniques Used: Binding Assay, Incubation, Staining

    35) Product Images from "Involvement of the P2X7 Purinergic Receptor in Colonic Motor Dysfunction Associated with Bowel Inflammation in Rats"

    Article Title: Involvement of the P2X7 Purinergic Receptor in Colonic Motor Dysfunction Associated with Bowel Inflammation in Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116253

    Double-staining immunohistochemistry showing the distribution of P2X7 receptors (green) and the neuronal marker HuC/D (red) in the myenteric plexus of colonic cryosections from control (A; normal) or DNBS-treated (B; colitis) rats. Nuclei were stained with TOTO-3. Scale bar: 21 µm. Enlarged view of HuC/D + and P2X7 + cells in the myenteric ganglia of normal and colitis rats from boxed area in overlay (scale bar = 10 µm). LM, longitudinal muscle; CM, circular muscle; MG, myenteric ganglia. Isotype fluorescent image was obtained by labeling with streptavidin conjugated with Alexa Fluor 555 in presence of normal mouse antiserum instead of the primary antibody.
    Figure Legend Snippet: Double-staining immunohistochemistry showing the distribution of P2X7 receptors (green) and the neuronal marker HuC/D (red) in the myenteric plexus of colonic cryosections from control (A; normal) or DNBS-treated (B; colitis) rats. Nuclei were stained with TOTO-3. Scale bar: 21 µm. Enlarged view of HuC/D + and P2X7 + cells in the myenteric ganglia of normal and colitis rats from boxed area in overlay (scale bar = 10 µm). LM, longitudinal muscle; CM, circular muscle; MG, myenteric ganglia. Isotype fluorescent image was obtained by labeling with streptavidin conjugated with Alexa Fluor 555 in presence of normal mouse antiserum instead of the primary antibody.

    Techniques Used: Double Staining Immunohistochemistry, Marker, Staining, Labeling

    36) Product Images from "Micropatterning Alginate Substrates for in vitro Cardiovascular Muscle on a Chip **"

    Article Title: Micropatterning Alginate Substrates for in vitro Cardiovascular Muscle on a Chip **

    Journal: Advanced functional materials

    doi: 10.1002/adfm.201203319

    Micropatterned alginate thin films fabricated using (A, B) microcontact printing and (C, D) micromolding. (A, C i) A layer of APTES is deposited on the glass coverslip (A, C ii–iii) A flat or patterned calcium-loaded agar stamp is applied on a drop of alginate (A, C iv) The thin film of hydrogel is submerged in a solution of streptavidin mixed with the reagents EDC and Sulfo-NHS and then washed and dried (A, C v) Biotinylated fibronectin is applied either by microcontact printing with a stamp of PDMS or by simple submersion (A, C vi) Samples are ready for being cut for contractility assay. (B i) Fluorescent imaging of immunostained 2D fibronectin pattern on flat alginate films (B ii) Section profile of fluorescence level. (D i) 3D reconstruction of AFM imaging of the topography of micromolded films (D ii) Height profile along a section perpendicular to the features.
    Figure Legend Snippet: Micropatterned alginate thin films fabricated using (A, B) microcontact printing and (C, D) micromolding. (A, C i) A layer of APTES is deposited on the glass coverslip (A, C ii–iii) A flat or patterned calcium-loaded agar stamp is applied on a drop of alginate (A, C iv) The thin film of hydrogel is submerged in a solution of streptavidin mixed with the reagents EDC and Sulfo-NHS and then washed and dried (A, C v) Biotinylated fibronectin is applied either by microcontact printing with a stamp of PDMS or by simple submersion (A, C vi) Samples are ready for being cut for contractility assay. (B i) Fluorescent imaging of immunostained 2D fibronectin pattern on flat alginate films (B ii) Section profile of fluorescence level. (D i) 3D reconstruction of AFM imaging of the topography of micromolded films (D ii) Height profile along a section perpendicular to the features.

    Techniques Used: Imaging, Fluorescence

    37) Product Images from "MIS416 as a siRNA Delivery System with the Ability to Target Antigen-Presenting Cells"

    Article Title: MIS416 as a siRNA Delivery System with the Ability to Target Antigen-Presenting Cells

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2017.0695

    Internalization of MIS416-PE (MIS416-biotin-streptavidin-PE) by splenocytes, and BMDCs. (A–C) Graphs showing internalization of MIS416-PE after 1, 4, or 24 h, respectively, at 37°C. Splenocytes were pulsed with MIS416-PE (1, 5, or 10 μg) for 1, 4, or 24 h. After incubation, cells were washed in PBS, and then immunocytochemically stained with five different antibodies in PBS (LY6, B220, CD11c, F480, CD3) to identify five different cell populations (neutrophils, B cells, DCs, macrophages, and T cells), respectively. The gating strategies are explained in Supplementary Fig. S1 . Error bars represent standard error of the mean (SEM). ns, not significant; * P
    Figure Legend Snippet: Internalization of MIS416-PE (MIS416-biotin-streptavidin-PE) by splenocytes, and BMDCs. (A–C) Graphs showing internalization of MIS416-PE after 1, 4, or 24 h, respectively, at 37°C. Splenocytes were pulsed with MIS416-PE (1, 5, or 10 μg) for 1, 4, or 24 h. After incubation, cells were washed in PBS, and then immunocytochemically stained with five different antibodies in PBS (LY6, B220, CD11c, F480, CD3) to identify five different cell populations (neutrophils, B cells, DCs, macrophages, and T cells), respectively. The gating strategies are explained in Supplementary Fig. S1 . Error bars represent standard error of the mean (SEM). ns, not significant; * P

    Techniques Used: Incubation, Staining

    38) Product Images from "Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes"

    Article Title: Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes

    Journal: eLife

    doi: 10.7554/eLife.19891

    CTxB clusters are not highly correlated with BCR. Average cross-correlation between clustered CTxB and BCR indicates that these two proteins are not colocalized. Biotinylated CTxB was clustered by streptavidin conjugated to Atto 655 and cells were fixed prior to labeling BCR with a f(Ab) 1 fragment conjugated to Alexa 532. The lack of pronounced cross-correlation between BCR and clustered CTxB indicates that CTxB is not forcing BCR to be clustered nor was it strongly recruiting BCR. Interestingly, the weak enrichment of BCR within CTxB clusters is similar to the enrichment of membrane anchored probes around clustered CTxB and BCR, suggesting that the enrichment stems from domain partitioning. The curve is an average of 6 cells with errorbars showing the standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.19891.027
    Figure Legend Snippet: CTxB clusters are not highly correlated with BCR. Average cross-correlation between clustered CTxB and BCR indicates that these two proteins are not colocalized. Biotinylated CTxB was clustered by streptavidin conjugated to Atto 655 and cells were fixed prior to labeling BCR with a f(Ab) 1 fragment conjugated to Alexa 532. The lack of pronounced cross-correlation between BCR and clustered CTxB indicates that CTxB is not forcing BCR to be clustered nor was it strongly recruiting BCR. Interestingly, the weak enrichment of BCR within CTxB clusters is similar to the enrichment of membrane anchored probes around clustered CTxB and BCR, suggesting that the enrichment stems from domain partitioning. The curve is an average of 6 cells with errorbars showing the standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.19891.027

    Techniques Used: Labeling

    Representative images from Figure 1 . Representative images from conditions included in average curves but not shown in Figure 1 . Scale bars are 5 µm. (left) Cells expressing mEos3.2-TM were labeled with CTxB-biotin that was then clustered with streptavidin-Atto655. (right) Cells expressing both mEos3.2-GG and YFP-TM. TM was clustered using a biotinylated anti-GFP antibody followed by streptavidin-Atto655. DOI: http://dx.doi.org/10.7554/eLife.19891.010
    Figure Legend Snippet: Representative images from Figure 1 . Representative images from conditions included in average curves but not shown in Figure 1 . Scale bars are 5 µm. (left) Cells expressing mEos3.2-TM were labeled with CTxB-biotin that was then clustered with streptavidin-Atto655. (right) Cells expressing both mEos3.2-GG and YFP-TM. TM was clustered using a biotinylated anti-GFP antibody followed by streptavidin-Atto655. DOI: http://dx.doi.org/10.7554/eLife.19891.010

    Techniques Used: Expressing, Labeling

    Representative images from Figure 2 . Representative images from conditions included in average curves but for which images are not shown in Figure 2 . Scale bars are 5 µm. In all images, IgM BCR is labeled with f(Ab) 1 conjugated to Atto655 and clustered with streptavidin. TM is expressed as a mEos3.2 fusion protein. From left to right: CH27 cell fixed 1 min following antigen addition; CH27 cell fixed 5 min following antigen addition; live CH27 cell reconstructed from frames following antigen addition; murine primary B cell fixed 5 min following antigen addition. DOI: http://dx.doi.org/10.7554/eLife.19891.021
    Figure Legend Snippet: Representative images from Figure 2 . Representative images from conditions included in average curves but for which images are not shown in Figure 2 . Scale bars are 5 µm. In all images, IgM BCR is labeled with f(Ab) 1 conjugated to Atto655 and clustered with streptavidin. TM is expressed as a mEos3.2 fusion protein. From left to right: CH27 cell fixed 1 min following antigen addition; CH27 cell fixed 5 min following antigen addition; live CH27 cell reconstructed from frames following antigen addition; murine primary B cell fixed 5 min following antigen addition. DOI: http://dx.doi.org/10.7554/eLife.19891.021

    Techniques Used: Labeling

    Cross-correlations between PM and unclustered BCR or CTxB are near detection limits. Representative images (top) showing cells expressing PM-mEos3.2 and labeled with Atto655 anti-IgM f(Ab) 1 (left) or Atto655-CTxB (right), where BCR or CTxB labels are not clustered with streptavidin. Scale bars are 5 µm. Average cross-correlations, C(r) (bottom), between unclustered BCR and lipid probes (left) and unclustered CTxB and lipid probes (right). Curves indicate an average over multiple cells (N): BCR and PM (14), CTxB and PM (18). Subtle enrichment of PM is evident in both cases. DOI: http://dx.doi.org/10.7554/eLife.19891.020
    Figure Legend Snippet: Cross-correlations between PM and unclustered BCR or CTxB are near detection limits. Representative images (top) showing cells expressing PM-mEos3.2 and labeled with Atto655 anti-IgM f(Ab) 1 (left) or Atto655-CTxB (right), where BCR or CTxB labels are not clustered with streptavidin. Scale bars are 5 µm. Average cross-correlations, C(r) (bottom), between unclustered BCR and lipid probes (left) and unclustered CTxB and lipid probes (right). Curves indicate an average over multiple cells (N): BCR and PM (14), CTxB and PM (18). Subtle enrichment of PM is evident in both cases. DOI: http://dx.doi.org/10.7554/eLife.19891.020

    Techniques Used: Expressing, Labeling

    Cell surface clustering of cholera toxin subunit B elicits calcium mobilization in B cells. Cytosolic calcium levels were monitored in CH27 B cells both before and after biotinylated CTxB was clustered with streptavidin using the calcium indicator Fluo-4 as described in Methods. Colored curves represent raw fluorescence intensity traces for single cells and the average response of 40 cells is shown in black. The blue shaded region denotes +/- one standard deviation between the averaged cells. One reason for the broad width of this distribution is that individual cells oscillate between high and low fluorescent states, as apparent in the single cell traces. DOI: http://dx.doi.org/10.7554/eLife.19891.026
    Figure Legend Snippet: Cell surface clustering of cholera toxin subunit B elicits calcium mobilization in B cells. Cytosolic calcium levels were monitored in CH27 B cells both before and after biotinylated CTxB was clustered with streptavidin using the calcium indicator Fluo-4 as described in Methods. Colored curves represent raw fluorescence intensity traces for single cells and the average response of 40 cells is shown in black. The blue shaded region denotes +/- one standard deviation between the averaged cells. One reason for the broad width of this distribution is that individual cells oscillate between high and low fluorescent states, as apparent in the single cell traces. DOI: http://dx.doi.org/10.7554/eLife.19891.026

    Techniques Used: Fluorescence, Standard Deviation

    39) Product Images from "Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters"

    Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04991-2

    Multiple HSET motors conjugated to quantum dots drive self-assembly of GMPCPP-MTs into asters. a EGFP-HSET or EGFP-HSETΔTail was conjugated to streptavidin-QDots via the N-terminal 6× His-tag and a biotin anti-His antibody at a 3:1 ratio and visualized via TIRF. Representative kymographs of EGFP-HSET-QDots (left, 1 nM EGFP-HSET: 0.33 nM QDot) and EGFP-HSETΔTail-QDots (right, 0.5 nM EGFP-HSETΔTail: 0.17 nM QDot) are shown ( x -scale, distance, 5 µm; y -scale, time, 10 s). b – d Velocities ( b ), run lengths ( c ), and end dwell times ( d ) for the indicated constructed conjugated to QDots at a 3:1 ratio (EGFP-HSET, black, EGFP-HSETΔTail, red) were determined by kymograph analysis and plotted as histograms for the population. Data are reported as the mean values (insets) from CDF fitting ± the 95% CI from bootstrapping for the indicated n particles from 2 independent experiments, where N ≥ 4 movies for each condition. Populations for EGFP-HSET-QDots (black, upper) and EGFP-HSETΔTail-QDots (red, lower) are shown. For run length/end dwell times, particles reaching the end of MTs/dissociating immediately (
    Figure Legend Snippet: Multiple HSET motors conjugated to quantum dots drive self-assembly of GMPCPP-MTs into asters. a EGFP-HSET or EGFP-HSETΔTail was conjugated to streptavidin-QDots via the N-terminal 6× His-tag and a biotin anti-His antibody at a 3:1 ratio and visualized via TIRF. Representative kymographs of EGFP-HSET-QDots (left, 1 nM EGFP-HSET: 0.33 nM QDot) and EGFP-HSETΔTail-QDots (right, 0.5 nM EGFP-HSETΔTail: 0.17 nM QDot) are shown ( x -scale, distance, 5 µm; y -scale, time, 10 s). b – d Velocities ( b ), run lengths ( c ), and end dwell times ( d ) for the indicated constructed conjugated to QDots at a 3:1 ratio (EGFP-HSET, black, EGFP-HSETΔTail, red) were determined by kymograph analysis and plotted as histograms for the population. Data are reported as the mean values (insets) from CDF fitting ± the 95% CI from bootstrapping for the indicated n particles from 2 independent experiments, where N ≥ 4 movies for each condition. Populations for EGFP-HSET-QDots (black, upper) and EGFP-HSETΔTail-QDots (red, lower) are shown. For run length/end dwell times, particles reaching the end of MTs/dissociating immediately (

    Techniques Used: Construct

    40) Product Images from "An integrated experimental and modeling approach to propose biotinylated PLGA microparticles as versatile targeting vehicles for drug delivery"

    Article Title: An integrated experimental and modeling approach to propose biotinylated PLGA microparticles as versatile targeting vehicles for drug delivery

    Journal: Progress in Biomaterials

    doi: 10.1186/2194-0517-2-3

    Fluorescent imaging confirms presence of biotin on the outside of microparticles. The presence and bioavailability of biotin on the surface of the PLGA microparticles was confirmed by incubating biotinylated microparticles with streptavidin-488. The particles were washed several times prior to visualization under the fluorescent microscope. Left image, Alexa 488 + biotin-PLGA microparticles. The presence of the 488 fluorophore (white) indicates the biotin was present and capable of attaching the steptavidin. Right image, Alexa 488 + PLGA microparticles.
    Figure Legend Snippet: Fluorescent imaging confirms presence of biotin on the outside of microparticles. The presence and bioavailability of biotin on the surface of the PLGA microparticles was confirmed by incubating biotinylated microparticles with streptavidin-488. The particles were washed several times prior to visualization under the fluorescent microscope. Left image, Alexa 488 + biotin-PLGA microparticles. The presence of the 488 fluorophore (white) indicates the biotin was present and capable of attaching the steptavidin. Right image, Alexa 488 + PLGA microparticles.

    Techniques Used: Imaging, Microscopy

    41) Product Images from "CD200 modulates macrophage cytokine secretion and phagocytosis in response to poly(lactic co-glycolic acid) microparticles and films"

    Article Title: CD200 modulates macrophage cytokine secretion and phagocytosis in response to poly(lactic co-glycolic acid) microparticles and films

    Journal: Journal of materials chemistry. B, Materials for biology and medicine

    doi: 10.1039/C6TB02269C

    phagocytosis of cd200-coated plga microparticles by bmdm. (a) representative fluorescence images of bmdm incubated with unmodified, streptavidin-, and cd200-plga microparticles (green), stained with phalloidin (red), hoechst (blue). scale bar is 20 μm. (b) representative flow cytometry histogram showing fluorescein intensity of bmdm incubated with different plga microparticles. (c) graph of % phagocytosis of particles by bmdm (left). each distinct symbol (●,■,▲) represents a separate biological replicate (independent experiment, each performed in duplicate). graph of phagocytosis of different particles, relative to unmodified plga particles (right). (d) graph of median fluorescein intensity of bmdm treated with different particles, relative to unmodified plga. (e) graph of median fluorescein intensity of phagocytosis+ bmdm in different particle conditions, relative to unmodified plga. data represents mean ± sem. n=3. *p
    Figure Legend Snippet: phagocytosis of cd200-coated plga microparticles by bmdm. (a) representative fluorescence images of bmdm incubated with unmodified, streptavidin-, and cd200-plga microparticles (green), stained with phalloidin (red), hoechst (blue). scale bar is 20 μm. (b) representative flow cytometry histogram showing fluorescein intensity of bmdm incubated with different plga microparticles. (c) graph of % phagocytosis of particles by bmdm (left). each distinct symbol (●,■,▲) represents a separate biological replicate (independent experiment, each performed in duplicate). graph of phagocytosis of different particles, relative to unmodified plga particles (right). (d) graph of median fluorescein intensity of bmdm treated with different particles, relative to unmodified plga. (e) graph of median fluorescein intensity of phagocytosis+ bmdm in different particle conditions, relative to unmodified plga. data represents mean ± sem. n=3. *p

    Techniques Used: Fluorescence, Incubation, Staining, Flow Cytometry, Cytometry

    phagocytosis of cd200-coated plga microparticles by human monocytes. (a) representative fluorescence images of human monocytes incubated with unmodified, streptavidin-, and cd200-plga microparticles (green), stained with dii (red), and hoechst (blue). scale bar is 20 μm. (b) representative flow cytometry histogram showing fluorescein intensity of human monocytes incubated with different plga microparticles. (c) graph of % phagocytosis of particles by human monocytes. (left). each distinct symbol (●,■,▲) represents a separate biological replicate (independent experiment, each performed in duplicate). graph of monocyte phagocytosis of different particles, relative to unmodified plga condition (right). (d) graph of median fluorescein intensity of human monocytes treated with different particles, relative to unmodified plga. (e) graph of median fluorescein intensity of phagocytosis+ human monocytes treated with different particle conditions, relative to unmodified plga. data represents mean ± sem. n=3. *p
    Figure Legend Snippet: phagocytosis of cd200-coated plga microparticles by human monocytes. (a) representative fluorescence images of human monocytes incubated with unmodified, streptavidin-, and cd200-plga microparticles (green), stained with dii (red), and hoechst (blue). scale bar is 20 μm. (b) representative flow cytometry histogram showing fluorescein intensity of human monocytes incubated with different plga microparticles. (c) graph of % phagocytosis of particles by human monocytes. (left). each distinct symbol (●,■,▲) represents a separate biological replicate (independent experiment, each performed in duplicate). graph of monocyte phagocytosis of different particles, relative to unmodified plga condition (right). (d) graph of median fluorescein intensity of human monocytes treated with different particles, relative to unmodified plga. (e) graph of median fluorescein intensity of phagocytosis+ human monocytes treated with different particle conditions, relative to unmodified plga. data represents mean ± sem. n=3. *p

    Techniques Used: Fluorescence, Incubation, Staining, Flow Cytometry, Cytometry

    42) Product Images from "CD1b tetramers bind ?? T cell receptors to identify a mycobacterial glycolipid-reactive T cell repertoire in humans"

    Article Title: CD1b tetramers bind ?? T cell receptors to identify a mycobacterial glycolipid-reactive T cell repertoire in humans

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20110665

    CD1b tetramers stain human αβ T cells. (a) Bacterial GMM is formed by glucose linked at the 6-position to a mycolyl unit that contains two chiral centers, which are in the R configuration at positions 2 and 3 (2 R , 3 R ). (b) Tetramerizable CD1b monomers were used in plate-bound antigen presentation experiments to measure IL-2 release by the CD1b-restricted human T cell line LDN5 in response to C32 GMM loaded overnight at 37°C (mean + SEM). (c) CD1b was loaded with GMMs that are naturally formed with R configuration at C2 and C3 (R, R) or synthetic GMM prepared with an S configuration at C2 or C3 (2 R ,3 S +2 S ,3 R ) and complexed to streptavidin-labeled APC (tetramer APC) and tested for staining LDN5 T cells. (d) CD1b tetramers were then loaded with GMMs of the indicated average chain length (C32, C54, or C80) and tested for staining LDN5. MFI is mean fluorescence intensity. Data are representative of three or more experiments.
    Figure Legend Snippet: CD1b tetramers stain human αβ T cells. (a) Bacterial GMM is formed by glucose linked at the 6-position to a mycolyl unit that contains two chiral centers, which are in the R configuration at positions 2 and 3 (2 R , 3 R ). (b) Tetramerizable CD1b monomers were used in plate-bound antigen presentation experiments to measure IL-2 release by the CD1b-restricted human T cell line LDN5 in response to C32 GMM loaded overnight at 37°C (mean + SEM). (c) CD1b was loaded with GMMs that are naturally formed with R configuration at C2 and C3 (R, R) or synthetic GMM prepared with an S configuration at C2 or C3 (2 R ,3 S +2 S ,3 R ) and complexed to streptavidin-labeled APC (tetramer APC) and tested for staining LDN5 T cells. (d) CD1b tetramers were then loaded with GMMs of the indicated average chain length (C32, C54, or C80) and tested for staining LDN5. MFI is mean fluorescence intensity. Data are representative of three or more experiments.

    Techniques Used: Staining, Labeling, Fluorescence

    Tetramer staining proves a specific trimolecular interaction among CD1b, GMM, and the clonotypic TCR. (a) The LDN5 T cell clone was stained with unloaded CD1b tetramers or CD1b tetramers loaded with C32 GMM. Tetramers were preincubated with 10 µg/ml isotype control antibody or anti-CD1b antibody. (b) Soluble TCR-α chains with hexahistidine tags and -β chains with streptavidin tags were formed into soluble TCR dimers. (c) Loaded and unloaded tetramers were preincubated with fivefold molar excess of soluble recombinant T cell receptors derived from CD1a-restricted (sCD8-2) or CD1b-restricted (sLDN5) T cell lines. Data are representative of three or more experiments.
    Figure Legend Snippet: Tetramer staining proves a specific trimolecular interaction among CD1b, GMM, and the clonotypic TCR. (a) The LDN5 T cell clone was stained with unloaded CD1b tetramers or CD1b tetramers loaded with C32 GMM. Tetramers were preincubated with 10 µg/ml isotype control antibody or anti-CD1b antibody. (b) Soluble TCR-α chains with hexahistidine tags and -β chains with streptavidin tags were formed into soluble TCR dimers. (c) Loaded and unloaded tetramers were preincubated with fivefold molar excess of soluble recombinant T cell receptors derived from CD1a-restricted (sCD8-2) or CD1b-restricted (sLDN5) T cell lines. Data are representative of three or more experiments.

    Techniques Used: Staining, Recombinant, Derivative Assay

    43) Product Images from "DNA Hybridization Using Surface Plasmon-Coupled Emission"

    Article Title: DNA Hybridization Using Surface Plasmon-Coupled Emission

    Journal: Analytical chemistry

    doi: 10.1021/ac034881e

    a Figure not drawn to scale. BSA–streptavidin = 90 Å, and ssCy3–DNA = 70 Å.
    Figure Legend Snippet: a Figure not drawn to scale. BSA–streptavidin = 90 Å, and ssCy3–DNA = 70 Å.

    Techniques Used:

    44) Product Images from "Human Papillomavirus Integration: Analysis by Molecular Combing and Fiber-FISH"

    Article Title: Human Papillomavirus Integration: Analysis by Molecular Combing and Fiber-FISH

    Journal: Current protocols in microbiology

    doi: 10.1002/cpmc.61

    Placing and sealing a top coverslip over the fiber-containing coverslip A. Hybridization, blocking, and streptavidin/antibody incubations are all performed with a top coverslip overtop the fiber coverslip. To ensure the solution has been evenly wicked across the fiber coverslip, solutions are added to a clean coverslip and, by holding the bottom of the slide, the fiber coverslip is tapped on top. B. Sealing the fiber coverslip and top coverslip with rubber cement allows hybridization to be performed on a slide warmer rather than in a humid chamber. Use fresh rubber cement and cut a centimeter off the tip of a 1000 μl pipette tip for ease of pipetting. Excess rubber cement is not detrimental, but only a fine ribbon of cement is needed to completely seal the coverslips.
    Figure Legend Snippet: Placing and sealing a top coverslip over the fiber-containing coverslip A. Hybridization, blocking, and streptavidin/antibody incubations are all performed with a top coverslip overtop the fiber coverslip. To ensure the solution has been evenly wicked across the fiber coverslip, solutions are added to a clean coverslip and, by holding the bottom of the slide, the fiber coverslip is tapped on top. B. Sealing the fiber coverslip and top coverslip with rubber cement allows hybridization to be performed on a slide warmer rather than in a humid chamber. Use fresh rubber cement and cut a centimeter off the tip of a 1000 μl pipette tip for ease of pipetting. Excess rubber cement is not detrimental, but only a fine ribbon of cement is needed to completely seal the coverslips.

    Techniques Used: Hybridization, Blocking Assay, Transferring

    45) Product Images from "Agonist-Induced Changes in Substituted Cysteine Accessibility Reveal Dynamic Extracellular Structure of M3–M4 Loop of Glutamate Receptor GluR6"

    Article Title: Agonist-Induced Changes in Substituted Cysteine Accessibility Reveal Dynamic Extracellular Structure of M3–M4 Loop of Glutamate Receptor GluR6

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.19-02-00644.1999

    wt GluR6 shows significantly less cysteine-specific biotinylation than GluR6(S684C). A, B , Representative Western blot analyses of wt GluR6 and GluR6(S684C) after incubation of transfected cells with NHS-SS-biotin ( A ; primary amine-specific) or HPDP-biotin ( B ; cysteine-specific), as described in Materials and Methods. 1 , 2 , 4 , and 8 % represent the fraction of the total cell membrane isolate, and B represents the total protein precipitated by streptavidin-linked beads, loaded in each lane. C , For blots shown in A and B , band intensities were measured by densitometry from lanes containing 1, 2, 4, and 8% of the total membrane protein to generate a standard curve. From these curves, the amount of biotinylated GluR6 was calculated as a fraction of the total membrane GluR6. D , Bars represent data from n = 7–12 different experiments. #  Significant difference ( p
    Figure Legend Snippet: wt GluR6 shows significantly less cysteine-specific biotinylation than GluR6(S684C). A, B , Representative Western blot analyses of wt GluR6 and GluR6(S684C) after incubation of transfected cells with NHS-SS-biotin ( A ; primary amine-specific) or HPDP-biotin ( B ; cysteine-specific), as described in Materials and Methods. 1 , 2 , 4 , and 8 % represent the fraction of the total cell membrane isolate, and B represents the total protein precipitated by streptavidin-linked beads, loaded in each lane. C , For blots shown in A and B , band intensities were measured by densitometry from lanes containing 1, 2, 4, and 8% of the total membrane protein to generate a standard curve. From these curves, the amount of biotinylated GluR6 was calculated as a fraction of the total membrane GluR6. D , Bars represent data from n = 7–12 different experiments. #  Significant difference ( p

    Techniques Used: Western Blot, Incubation, Transfection

    46) Product Images from "A single-molecule flow platform for the quantification of biomolecules attached to single nanoparticles"

    Article Title: A single-molecule flow platform for the quantification of biomolecules attached to single nanoparticles

    Journal: Analytical chemistry

    doi: 10.1021/acs.analchem.8b00024

    Measurements obtained at the bulk and singlemolecule level. a) Fluorescence titration assays of varying numbers of biotin-Alexa647 bound to streptavidin. The control curve (red, without streptavidin) was measured in the absence of streptavidin. It shows
    Figure Legend Snippet: Measurements obtained at the bulk and singlemolecule level. a) Fluorescence titration assays of varying numbers of biotin-Alexa647 bound to streptavidin. The control curve (red, without streptavidin) was measured in the absence of streptavidin. It shows

    Techniques Used: Fluorescence, Titration

    Quantification of the number of streptavidin on individual Pdot-streptavidin (SA)-biotin-Alexa647 complexes. a,c) Experimental data (black line) and best-fit results (red line); the dotted blue line is a plot of the residuals of the fit compared to the
    Figure Legend Snippet: Quantification of the number of streptavidin on individual Pdot-streptavidin (SA)-biotin-Alexa647 complexes. a,c) Experimental data (black line) and best-fit results (red line); the dotted blue line is a plot of the residuals of the fit compared to the

    Techniques Used:

    Quantification of the number of streptavidin on the surface of Pdot-streptavidin (SA) conjugates. a) Schematic showing the labelling and measurement procedures. Signal 1 from APD1 is from PFO Pdots with blue fluorescence emission; signal 2 from APD2 is
    Figure Legend Snippet: Quantification of the number of streptavidin on the surface of Pdot-streptavidin (SA) conjugates. a) Schematic showing the labelling and measurement procedures. Signal 1 from APD1 is from PFO Pdots with blue fluorescence emission; signal 2 from APD2 is

    Techniques Used: Fluorescence

    Fluorescence intensity distributions of singly and fully labeled Pdot-streptavidin (SA)-biotin-Alexa647 complexes. a) Determination of the concentration ratios of streptavidin to Pdot to produce singly (green arrows) and fully (blue arrows) streptavidin-labeled
    Figure Legend Snippet: Fluorescence intensity distributions of singly and fully labeled Pdot-streptavidin (SA)-biotin-Alexa647 complexes. a) Determination of the concentration ratios of streptavidin to Pdot to produce singly (green arrows) and fully (blue arrows) streptavidin-labeled

    Techniques Used: Fluorescence, Labeling, Concentration Assay

    47) Product Images from "A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression"

    Article Title: A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20060136

    CyP inhibits LPS at the level of MD-2–TLR4 extracellular domain. (A) Luciferase activity of Jurkat cells transfected with a 3×NF-κB–driven luciferase reporter together with empty vector (Mock) or expression vectors encoding either TLR4, TLR9, or a chimera of extracellular TLR9 and intracellular TLR4 (TLR9N4C) and stimulated with 1 μg/ml LPS or 3 μM CpG. Reporter activity was measured after 16 h of LPS stimulation in the absence (white bars) or presence (black bars) of 20 μg/ml CyP. Similar results were obtained at 6 h of stimulation. Data represent the mean ± SD of duplicates of one experiment of two performed with identical results. (B) Spontaneous luciferase activity in Jurkat cells transfected with a 3×NF-κB–driven luciferase reporter together with an empty vector (Mock) or an expression vector encoding TLR4. Reporter activity in the absence (white bars) or presence (black bars) of CyP was measured 40 h after transfection in a 6-h assay. (C) Monocytes were stained with LPS conjugated to Alexa Fluor 488 (0.25 μg/ml LPS-AF488) in the absence (thick line) or presence (thin lines) of increasing concentrations of CyP (0.25, 12.5, 125, and 250 μg/ml) and analyzed by FACS. Background fluorescence of monocytes is shown as a gray profile. One representative experiment of four is shown. Fig. S3 shows the EC50 of CyP inhibiting LPS binding. (D) HEK293T cells mock transfected or transfected with MD-2–FLAG were either lysed and probed for MD-2 expression with anti-FLAG antibodies or treated with 20 μg/ml biotinylated CyP. Biotinylated CyP was then captured with immobilized streptavidin, and MD-2 coprecipitates were detected with anti-FLAG antibodies. Stripped blots were subsequently probed with anti–MD-2 antibodies. Arrows indicate specific bands of differentially glycosylated MD-2 (reference 63 ). (E) Recombinant human MD-2 (1 μg/ml, fixed concentration) was incubated in wells coated with CyP (left) or LPS (right) in the presence of increasing concentrations of soluble LPS or CyP (0.24, 0.74, 2.22, 6.66, 20, and 60 μg/ml). MD-2 bound to the coated plate was then detected by a specific anti–MD-2 antibody followed by horseradish peroxidase–conjugated secondary antibody. EC50 values were calculated on sigmoidal dose–response curves (variable slope; R squared, 0.9953 or 0.9939 and 0.9692 or 0.9996 for CyP and LPS on CyP or LPS coat, respectively). Data shown are from one experiment of two performed with identical results.
    Figure Legend Snippet: CyP inhibits LPS at the level of MD-2–TLR4 extracellular domain. (A) Luciferase activity of Jurkat cells transfected with a 3×NF-κB–driven luciferase reporter together with empty vector (Mock) or expression vectors encoding either TLR4, TLR9, or a chimera of extracellular TLR9 and intracellular TLR4 (TLR9N4C) and stimulated with 1 μg/ml LPS or 3 μM CpG. Reporter activity was measured after 16 h of LPS stimulation in the absence (white bars) or presence (black bars) of 20 μg/ml CyP. Similar results were obtained at 6 h of stimulation. Data represent the mean ± SD of duplicates of one experiment of two performed with identical results. (B) Spontaneous luciferase activity in Jurkat cells transfected with a 3×NF-κB–driven luciferase reporter together with an empty vector (Mock) or an expression vector encoding TLR4. Reporter activity in the absence (white bars) or presence (black bars) of CyP was measured 40 h after transfection in a 6-h assay. (C) Monocytes were stained with LPS conjugated to Alexa Fluor 488 (0.25 μg/ml LPS-AF488) in the absence (thick line) or presence (thin lines) of increasing concentrations of CyP (0.25, 12.5, 125, and 250 μg/ml) and analyzed by FACS. Background fluorescence of monocytes is shown as a gray profile. One representative experiment of four is shown. Fig. S3 shows the EC50 of CyP inhibiting LPS binding. (D) HEK293T cells mock transfected or transfected with MD-2–FLAG were either lysed and probed for MD-2 expression with anti-FLAG antibodies or treated with 20 μg/ml biotinylated CyP. Biotinylated CyP was then captured with immobilized streptavidin, and MD-2 coprecipitates were detected with anti-FLAG antibodies. Stripped blots were subsequently probed with anti–MD-2 antibodies. Arrows indicate specific bands of differentially glycosylated MD-2 (reference 63 ). (E) Recombinant human MD-2 (1 μg/ml, fixed concentration) was incubated in wells coated with CyP (left) or LPS (right) in the presence of increasing concentrations of soluble LPS or CyP (0.24, 0.74, 2.22, 6.66, 20, and 60 μg/ml). MD-2 bound to the coated plate was then detected by a specific anti–MD-2 antibody followed by horseradish peroxidase–conjugated secondary antibody. EC50 values were calculated on sigmoidal dose–response curves (variable slope; R squared, 0.9953 or 0.9939 and 0.9692 or 0.9996 for CyP and LPS on CyP or LPS coat, respectively). Data shown are from one experiment of two performed with identical results.

    Techniques Used: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Staining, FACS, Fluorescence, Binding Assay, Recombinant, Concentration Assay, Incubation

    48) Product Images from "Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces"

    Article Title: Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

    Journal: ACS applied materials & interfaces

    doi: 10.1021/am2009597

    One-component and three-component patterns of biomolecules on BP-modified substrates. (A) Photoimmobilization of biotinylated ConA in the “Illinois logo” pattern, visualized with fluorescently labeled streptavidin. (B) Three-component
    Figure Legend Snippet: One-component and three-component patterns of biomolecules on BP-modified substrates. (A) Photoimmobilization of biotinylated ConA in the “Illinois logo” pattern, visualized with fluorescently labeled streptavidin. (B) Three-component

    Techniques Used: Modification, Labeling

    49) Product Images from "Role of the Terminal Domains in Sodium Channel Localization"

    Article Title: Role of the Terminal Domains in Sodium Channel Localization

    Journal: Channels (Austin, Tex.)

    doi:

    Na v 1.2 Sorts to the Apical Membrane of MDCK Cells A. Na v 1.2-FLAG proteins expressed in the plasma membrane were isolated from the lysates of apically or basolaterally biotinylated MDCK cells stably expressing Na v 1.2-FLAG. Na v 1.2-FLAG proteins were first collected using antibodies against the FLAG epitope, and then the biotinylated Na v 1.2-FLAG proteins were precipitated from the Na v 1.2-FLAG pool using streptavidin. The final isolate was analyzed by SDS-PAGE and Western blot using anti-FLAG antibody. Isolate from 2.7×10 5 cells was loaded in each lane. B. Na v 1.6-myc proteins were similarly collected from MDCK cells stably expressing Na v 1.6-myc and analyzed, except anti-myc antibody was used in place of the anti-FLAG antibody. Isolate from 1.1×10 6 cells was loaded in each lane. C. MDCK cells stably expressing Na v 1.6-myc were fixed, permeabilized, and probed with anti-myc antibody. Myc staining was absent from cell-to-cell membrane junctions (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: Na v 1.2 Sorts to the Apical Membrane of MDCK Cells A. Na v 1.2-FLAG proteins expressed in the plasma membrane were isolated from the lysates of apically or basolaterally biotinylated MDCK cells stably expressing Na v 1.2-FLAG. Na v 1.2-FLAG proteins were first collected using antibodies against the FLAG epitope, and then the biotinylated Na v 1.2-FLAG proteins were precipitated from the Na v 1.2-FLAG pool using streptavidin. The final isolate was analyzed by SDS-PAGE and Western blot using anti-FLAG antibody. Isolate from 2.7×10 5 cells was loaded in each lane. B. Na v 1.6-myc proteins were similarly collected from MDCK cells stably expressing Na v 1.6-myc and analyzed, except anti-myc antibody was used in place of the anti-FLAG antibody. Isolate from 1.1×10 6 cells was loaded in each lane. C. MDCK cells stably expressing Na v 1.6-myc were fixed, permeabilized, and probed with anti-myc antibody. Myc staining was absent from cell-to-cell membrane junctions (arrowheads). Scale bars, 10 μm.

    Techniques Used: Isolation, Stable Transfection, Expressing, FLAG-tag, SDS Page, Western Blot, Staining

    50) Product Images from "Bioactivation and Cell Targeting of Semiconductor CdSe/ZnS Nanocrystals with Phytochelatin-Related Peptides"

    Article Title: Bioactivation and Cell Targeting of Semiconductor CdSe/ZnS Nanocrystals with Phytochelatin-Related Peptides

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja031691c

    Schematic representation of apparent steric hindrance problems associated with ligand density on the nanocrystal surface. Nanocrystals coated only with biotinylated peptides (A) have difficulty binding strepta-vidin bound to a surface probably because of the surface molecular crowding or high density of ligands. Reducing the amount of surface biotin using 1:1 mixtures of biotinylated peptides and nonbiotinylated peptides of similar length (B) does not improve the binding efficiency, the surface being still too crowded. When smaller peptides such as the PEG-modified peptides 9 are mixed with the biotinylated peptides (C) the resulting better exposure of biotin enables efficient binding to streptavidin on surface.
    Figure Legend Snippet: Schematic representation of apparent steric hindrance problems associated with ligand density on the nanocrystal surface. Nanocrystals coated only with biotinylated peptides (A) have difficulty binding strepta-vidin bound to a surface probably because of the surface molecular crowding or high density of ligands. Reducing the amount of surface biotin using 1:1 mixtures of biotinylated peptides and nonbiotinylated peptides of similar length (B) does not improve the binding efficiency, the surface being still too crowded. When smaller peptides such as the PEG-modified peptides 9 are mixed with the biotinylated peptides (C) the resulting better exposure of biotin enables efficient binding to streptavidin on surface.

    Techniques Used: Binding Assay, Modification

    Streptavidin gel retardation assay with biotinylated peptide-coated nanocrystals. (a) 1% agarose gel electrophoresis of biotinylated red emitting CdSe/ZnS nanocrystals coated with peptide 8 . The incubation of the nanocrystals with increasing concentration of streptavidin leads to the formation of aggregates and a shift of the nanoparticle band. Large nanocrystal aggregates are unable to enter the gel pores. The gel re-entry at high streptavidin concentration corresponds to the saturation of the nanocrystal surface biotins that prevents aggregation. (b) 1% agarose electrophoresis of nonbiotinylated CdSe/ZnS nanocrystals coated with peptide 6 under the same condition as in (a). The concentrations of streptavidin tested were 2000, 1000, 750, 500, 300, 250, 150, 100, 50, 40, 30, 25, 20, 15, 10, 5, 2 and 1 μ g/mL. *: no streptavidin; **: biotinylated nanocrystals + 250 μ g/mL streptavidin.
    Figure Legend Snippet: Streptavidin gel retardation assay with biotinylated peptide-coated nanocrystals. (a) 1% agarose gel electrophoresis of biotinylated red emitting CdSe/ZnS nanocrystals coated with peptide 8 . The incubation of the nanocrystals with increasing concentration of streptavidin leads to the formation of aggregates and a shift of the nanoparticle band. Large nanocrystal aggregates are unable to enter the gel pores. The gel re-entry at high streptavidin concentration corresponds to the saturation of the nanocrystal surface biotins that prevents aggregation. (b) 1% agarose electrophoresis of nonbiotinylated CdSe/ZnS nanocrystals coated with peptide 6 under the same condition as in (a). The concentrations of streptavidin tested were 2000, 1000, 750, 500, 300, 250, 150, 100, 50, 40, 30, 25, 20, 15, 10, 5, 2 and 1 μ g/mL. *: no streptavidin; **: biotinylated nanocrystals + 250 μ g/mL streptavidin.

    Techniques Used: Electrophoretic Mobility Shift Assay, Agarose Gel Electrophoresis, Incubation, Concentration Assay, Electrophoresis

    51) Product Images from "Ultrasound Molecular Imaging of Angiogenesis Using Vascular Endothelial Growth Factor-Conjugated Microbubbles"

    Article Title: Ultrasound Molecular Imaging of Angiogenesis Using Vascular Endothelial Growth Factor-Conjugated Microbubbles

    Journal: Molecular pharmaceutics

    doi: 10.1021/acs.molpharmaceut.6b01033

    (A) The average number of streptavidin molecules on the MB surface as a function of streptavidin incubation concentration. The saturation concentration at 0.25 mg/mL was used for maximum conjugation, corresponding to about 8×10 5 streptavidin molecules
    Figure Legend Snippet: (A) The average number of streptavidin molecules on the MB surface as a function of streptavidin incubation concentration. The saturation concentration at 0.25 mg/mL was used for maximum conjugation, corresponding to about 8×10 5 streptavidin molecules

    Techniques Used: Incubation, Concentration Assay, Conjugation Assay

    52) Product Images from "Identification of a mast cell specific receptor crucial for pseudo-allergic drug reactions"

    Article Title: Identification of a mast cell specific receptor crucial for pseudo-allergic drug reactions

    Journal: Nature

    doi: 10.1038/nature14022

    Human mast cells are activated by basic secretagogues and drugs associated with pseudo-allergic reactions in an MrgprX2-dependent manner. a . Human LAD2 mast cells were treated with different concentrations of compound 48/80, mastoparan, icatibant, atracurium, and ciprofloxacin. The activation of mast cells in response to these substances was characterized by the release of β-hexosaminidase, TNF, PGD2, and histamine. In addition, 0.1 μg/ml streptavidin stimulation of biotin-conjugated human IgE sensitized LAD2 cells caused a robust release of β-hexosaminidase (71.3±1.8% release), compared to untreated cells (4.1±0.3% release). Group data are expressed as mean ± standard error of the mean. b . Knockdown of human MrgprX2 significantly reduced mast cell activation evoked by basic secretagogues and drugs associated with pseudo-allergic reactions, but not by IgE. Human LAD2 mast cells were first transfected with MrgprX2 siRNA or control siRNA. Two days after the transfection, the cells were treated with compound 48/80 (0.1 μg/ml), mastoparan (5 μg/ml), icatibant (10 μg/ml), atracurium (25 μg/ml), and ciprofloxacin (75 μg/ml). The activation of mast cells in response to these substances characterized by the release of β-hexosaminidase was significantly reduced in MrgprX2 siRNA treated cells, compared to release in the control group. IgE-mediated mast cell degranulation was unaffected by MrgprX2 siRNA knockdown. Group data are expressed as mean ± standard error of the mean. Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at * p
    Figure Legend Snippet: Human mast cells are activated by basic secretagogues and drugs associated with pseudo-allergic reactions in an MrgprX2-dependent manner. a . Human LAD2 mast cells were treated with different concentrations of compound 48/80, mastoparan, icatibant, atracurium, and ciprofloxacin. The activation of mast cells in response to these substances was characterized by the release of β-hexosaminidase, TNF, PGD2, and histamine. In addition, 0.1 μg/ml streptavidin stimulation of biotin-conjugated human IgE sensitized LAD2 cells caused a robust release of β-hexosaminidase (71.3±1.8% release), compared to untreated cells (4.1±0.3% release). Group data are expressed as mean ± standard error of the mean. b . Knockdown of human MrgprX2 significantly reduced mast cell activation evoked by basic secretagogues and drugs associated with pseudo-allergic reactions, but not by IgE. Human LAD2 mast cells were first transfected with MrgprX2 siRNA or control siRNA. Two days after the transfection, the cells were treated with compound 48/80 (0.1 μg/ml), mastoparan (5 μg/ml), icatibant (10 μg/ml), atracurium (25 μg/ml), and ciprofloxacin (75 μg/ml). The activation of mast cells in response to these substances characterized by the release of β-hexosaminidase was significantly reduced in MrgprX2 siRNA treated cells, compared to release in the control group. IgE-mediated mast cell degranulation was unaffected by MrgprX2 siRNA knockdown. Group data are expressed as mean ± standard error of the mean. Two-tailed unpaired Student's t test was used to determine significance in statistical comparisons, and differences were considered significant at * p

    Techniques Used: Activation Assay, Transfection, Two Tailed Test

    53) Product Images from "Proteome-wide Mapping of Cholesterol-Interacting Proteins in Mammalian Cells"

    Article Title: Proteome-wide Mapping of Cholesterol-Interacting Proteins in Mammalian Cells

    Journal: Nature methods

    doi: 10.1038/nmeth.2368

    MS-based profiling of sterol-binding proteins in HeLa cells ( a ) Scheme for enrichment and analysis of sterol probe labeling profiles in mammalian cells by biotin-streptavidin methods and SILAC MS analysis. ( b–d ) Heavy/light ratio plots for total proteins identified in experiments that compared the labeling profiles of the trans -sterol probe versus no-UV light control ( b ; 20 μM trans probe / 20 μM trans probe with no UV), the PEA-DA probe ( c ; 20 μM trans probe / 20 μM PEA-DA probe), and 10× cholesterol competition ( d ; 10 μM trans probe / 10 μM trans probe + 100 μM cholesterol). Representative MS1 traces with calculated ratios for proteins that fall into Groups I-IV, as well as the MS1 traces for a non-specific background protein, are shown to the right of the global ratio plots. Ratios of > 20 are listed as 20. ( e ) Venn diagram showing the distribution of Group I-IV proteins for the trans -sterol probe labeling profile. ( f ) Top-five pathways determined by searching Group I proteins on the KEGG database, and top-12 biological function networks determined by searching Group I proteins on the DAVID gene ontology server. ( g ) Trans -sterol probe labeling profile for the cholesterol biosynthetic pathway, with colors reflecting each enzyme's Group designation (black: not detected).
    Figure Legend Snippet: MS-based profiling of sterol-binding proteins in HeLa cells ( a ) Scheme for enrichment and analysis of sterol probe labeling profiles in mammalian cells by biotin-streptavidin methods and SILAC MS analysis. ( b–d ) Heavy/light ratio plots for total proteins identified in experiments that compared the labeling profiles of the trans -sterol probe versus no-UV light control ( b ; 20 μM trans probe / 20 μM trans probe with no UV), the PEA-DA probe ( c ; 20 μM trans probe / 20 μM PEA-DA probe), and 10× cholesterol competition ( d ; 10 μM trans probe / 10 μM trans probe + 100 μM cholesterol). Representative MS1 traces with calculated ratios for proteins that fall into Groups I-IV, as well as the MS1 traces for a non-specific background protein, are shown to the right of the global ratio plots. Ratios of > 20 are listed as 20. ( e ) Venn diagram showing the distribution of Group I-IV proteins for the trans -sterol probe labeling profile. ( f ) Top-five pathways determined by searching Group I proteins on the KEGG database, and top-12 biological function networks determined by searching Group I proteins on the DAVID gene ontology server. ( g ) Trans -sterol probe labeling profile for the cholesterol biosynthetic pathway, with colors reflecting each enzyme's Group designation (black: not detected).

    Techniques Used: Mass Spectrometry, Binding Assay, Labeling

    54) Product Images from "Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch"

    Article Title: Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1712983115

    Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage T7 RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.
    Figure Legend Snippet: Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage T7 RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.

    Techniques Used: Fluorescence, Labeling, Incubation, Microscopy

    55) Product Images from "Oxidative stress-induced protein damage inhibits DNA repair and determines mutation risk and anticancer drug effectiveness"

    Article Title: Oxidative stress-induced protein damage inhibits DNA repair and determines mutation risk and anticancer drug effectiveness

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-16-0053

    UV-induced ROS, protein oxidation and excision repair A. Measurement of ROS in HaCaT cells irradiated with UVA, UVB or UVC. ROS were detected using the CM-H 2 CDFDA probe and FACS analysis. B. UV-induced protein carbonyls in HaCaT cells. UVA-, UVB- and UVC-irradiated HaCaT cell extracts prepared immediately after irradiation were derivatized with Hydroxylamine Alexa Fluor 647 and separated by PAGE. Carbonylated proteins were visualised at 633 nm. C. Protein sulfenates in HaCaT cells following UVA irradiation. Proteins in extracts prepared immediately after UVA irradiation were reacted with the biotin-tagged probe 1,3-cyclopentanedione (BP-1) and captured on streptavidin beads. Eluted proteins were separated by PAGE and transferred to membranes that were probed with streptavidin-HRP. D. Removal of UVB-induced 6:4 Py:Pys in UVA-irradiated HaCaT cells. Cells were irradiated with 200 J/m 2 UVB and UVA at the doses indicated. DNA was extracted at the times indicated. 6:4 Py:Pys were measured by ELISA. Data for UVB and UVB + 200 kJ/m 2 UVA are means of 9 and 6 determinations, respectively. Other data are the means of at least 2 independent experiments. E. Removal of CPDs by HaCaT cells. Cells were irradiated with UVA (300 kJ/m 2 ), UVB (300 J/m 2 ) or UVC (7 J/m 2 ) to induce equal numbers of CPDs. DNA was extracted at the times indicated. CPDs were measured by ELISA. The data are the means of 2 independent experiments. F. DNA 8-oxoG excision by HaCaT cells. Cells were treated with 2.5 mM KBrO 3 ± 200 kJ/m 2 UVA. DNA was extracted at the times indicated. DNA 8-oxoG was measured by ELISA. The data are the means of 2 independent experiments. G. H 2 O 2 -induced protein carbonyls in HaCaT cells. Cells were treated with 500 mM H 2 O 2 for 30 min. Extracted proteins were derivatized with Hydroxylamine Alexa Fluor 647 and separated by PAGE. Carbonyls were visualised at 633 nm H. H 2 O 2 -induced inhibition of NER. HaCaT cells treated as in A were irradiated with 200 J/m 2 UVB. DNA was extracted at the times indicated and 6:4 Py:Pys were measured by ELISA.
    Figure Legend Snippet: UV-induced ROS, protein oxidation and excision repair A. Measurement of ROS in HaCaT cells irradiated with UVA, UVB or UVC. ROS were detected using the CM-H 2 CDFDA probe and FACS analysis. B. UV-induced protein carbonyls in HaCaT cells. UVA-, UVB- and UVC-irradiated HaCaT cell extracts prepared immediately after irradiation were derivatized with Hydroxylamine Alexa Fluor 647 and separated by PAGE. Carbonylated proteins were visualised at 633 nm. C. Protein sulfenates in HaCaT cells following UVA irradiation. Proteins in extracts prepared immediately after UVA irradiation were reacted with the biotin-tagged probe 1,3-cyclopentanedione (BP-1) and captured on streptavidin beads. Eluted proteins were separated by PAGE and transferred to membranes that were probed with streptavidin-HRP. D. Removal of UVB-induced 6:4 Py:Pys in UVA-irradiated HaCaT cells. Cells were irradiated with 200 J/m 2 UVB and UVA at the doses indicated. DNA was extracted at the times indicated. 6:4 Py:Pys were measured by ELISA. Data for UVB and UVB + 200 kJ/m 2 UVA are means of 9 and 6 determinations, respectively. Other data are the means of at least 2 independent experiments. E. Removal of CPDs by HaCaT cells. Cells were irradiated with UVA (300 kJ/m 2 ), UVB (300 J/m 2 ) or UVC (7 J/m 2 ) to induce equal numbers of CPDs. DNA was extracted at the times indicated. CPDs were measured by ELISA. The data are the means of 2 independent experiments. F. DNA 8-oxoG excision by HaCaT cells. Cells were treated with 2.5 mM KBrO 3 ± 200 kJ/m 2 UVA. DNA was extracted at the times indicated. DNA 8-oxoG was measured by ELISA. The data are the means of 2 independent experiments. G. H 2 O 2 -induced protein carbonyls in HaCaT cells. Cells were treated with 500 mM H 2 O 2 for 30 min. Extracted proteins were derivatized with Hydroxylamine Alexa Fluor 647 and separated by PAGE. Carbonyls were visualised at 633 nm H. H 2 O 2 -induced inhibition of NER. HaCaT cells treated as in A were irradiated with 200 J/m 2 UVB. DNA was extracted at the times indicated and 6:4 Py:Pys were measured by ELISA.

    Techniques Used: Irradiation, FACS, Polyacrylamide Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Inhibition

    Protein damage NER in vitro and inhibition by 1 O 2 A. Nuclear extracts prepared from HeLa cells that had been irradiated as indicated were assayed for NER. NER excision products (indicated) were end-radiolabelled and separated by gel electrophoresis. B. GelDoc quantitation of excision. Means of NER assays with 3 independent extracts from control or UVA (200 kJ/m 2 ) irradiated HeLa cells. C. RPA32 sulfenates in UVA-treated HaCaT cells. Following derivatisation with BP-1 and streptavidin bead capture, proteins were recovered, separated by PAGE and immunoblots were probed for RPA32. Eluate = streptavidin-captured samples; input = samples prior to streptavidin bead loading. D. 1 O 2 and UVA-induced protein carbonyls in HaCaT cells. Cells were irradiated with 200 kJ/m 2 UVA in PBS prepared with H 2 O or D 2 O as indicated. Extracts were prepared and protein carbonyls were derivatized using Hydroxylamine Alexa Fluor 647, separated by PAGE and visualised at 633 nm. E. 1 O 2 and NER. HaCaT cells were irradiated with 200 J/m 2 UVB ± 200 kJ/m 2 UVA in PBS prepared with H 2 O or D 2 O. DNA was extracted at the times indicated and 6:4 Py:Pys measured by ELISA. D 2 O data represent the mean of 2 independent experiments.
    Figure Legend Snippet: Protein damage NER in vitro and inhibition by 1 O 2 A. Nuclear extracts prepared from HeLa cells that had been irradiated as indicated were assayed for NER. NER excision products (indicated) were end-radiolabelled and separated by gel electrophoresis. B. GelDoc quantitation of excision. Means of NER assays with 3 independent extracts from control or UVA (200 kJ/m 2 ) irradiated HeLa cells. C. RPA32 sulfenates in UVA-treated HaCaT cells. Following derivatisation with BP-1 and streptavidin bead capture, proteins were recovered, separated by PAGE and immunoblots were probed for RPA32. Eluate = streptavidin-captured samples; input = samples prior to streptavidin bead loading. D. 1 O 2 and UVA-induced protein carbonyls in HaCaT cells. Cells were irradiated with 200 kJ/m 2 UVA in PBS prepared with H 2 O or D 2 O as indicated. Extracts were prepared and protein carbonyls were derivatized using Hydroxylamine Alexa Fluor 647, separated by PAGE and visualised at 633 nm. E. 1 O 2 and NER. HaCaT cells were irradiated with 200 J/m 2 UVB ± 200 kJ/m 2 UVA in PBS prepared with H 2 O or D 2 O. DNA was extracted at the times indicated and 6:4 Py:Pys measured by ELISA. D 2 O data represent the mean of 2 independent experiments.

    Techniques Used: In Vitro, Inhibition, Irradiation, Nucleic Acid Electrophoresis, Quantitation Assay, Polyacrylamide Gel Electrophoresis, Western Blot, Enzyme-linked Immunosorbent Assay

    56) Product Images from "Activation of Estrogen Receptor-? by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale Chromatin Modification and mRNA Transcription"

    Article Title: Activation of Estrogen Receptor-? by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale Chromatin Modification and mRNA Transcription

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002286

    Temporal single-cell analyses of ER-S118A and ER-S118E transcriptional response at the PRL-array. PRL-HeLa cells transiently expressing the two phosphomutants, GFP-ERS118A (panel A and C) or GFP-ERS118E (panel B and D), were treated with either ethanol, E2 (panel A and B) or EGF (panel C and D) for different times. Subsequent to ligand treatment, the cells were fixed and subjected to RNA FISH using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. The FISH signal at the array was determined as described and the value graphed as fold induction over vehicle control cells (solid line). For each cell the area of the array was also determined and plotted as fold induction over vehicle control (dotted line). Data represent the mean ±SEM of three different experiments graphed as fold induction over time-matched vehicle-treated control cells.
    Figure Legend Snippet: Temporal single-cell analyses of ER-S118A and ER-S118E transcriptional response at the PRL-array. PRL-HeLa cells transiently expressing the two phosphomutants, GFP-ERS118A (panel A and C) or GFP-ERS118E (panel B and D), were treated with either ethanol, E2 (panel A and B) or EGF (panel C and D) for different times. Subsequent to ligand treatment, the cells were fixed and subjected to RNA FISH using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. The FISH signal at the array was determined as described and the value graphed as fold induction over vehicle control cells (solid line). For each cell the area of the array was also determined and plotted as fold induction over vehicle control (dotted line). Data represent the mean ±SEM of three different experiments graphed as fold induction over time-matched vehicle-treated control cells.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization

    ER transcriptional activity at the promoter array. PRL-HeLa cells transiently expressing GFP-ER were treated with E2, EGF, 4-hydroxytamoxifen (4HT) or ethanolic vehicle for the indicated time. Subsequent to ligand treatment the cells were fixed and subjected to an RNA FISH protocol using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. A. Representative images of a single cell for each treatment. The presence of transcripts at the promoter array is identified by accumulated signal above the level for the nucleoplasm. The inset values (red type) represent the amount of transcript at 2 hours, relative to vehicle controls. B. Representative image of PRL-HeLa cells transfected with GFP-ER (green) exemplify the heterogeneity of ER expression levels. The cells show the RNA FISH signal associated with the array in the cell population (red signal) (I). The nuclear GFP-ER mean of fluorescence and RNA FISH array signal were plotted for vehicle- and E2-treated cells. Each symbol represents the measurements from a singe cell (II). C. To quantify FISH signal over 24 hours the total intensity of signal at the array (minus background signal) was determined by cumulative summation of 20 planes. Data represent the mean ±SEM of three different experiments graphed as fold induction over mean time-matched vehicle-treated control cells. D. This panel represents the earlier time points for E2 and EGF treatment. Fold activation between EGF and E2 at 30 minutes was significantly different, with a p value of 0.03.
    Figure Legend Snippet: ER transcriptional activity at the promoter array. PRL-HeLa cells transiently expressing GFP-ER were treated with E2, EGF, 4-hydroxytamoxifen (4HT) or ethanolic vehicle for the indicated time. Subsequent to ligand treatment the cells were fixed and subjected to an RNA FISH protocol using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. A. Representative images of a single cell for each treatment. The presence of transcripts at the promoter array is identified by accumulated signal above the level for the nucleoplasm. The inset values (red type) represent the amount of transcript at 2 hours, relative to vehicle controls. B. Representative image of PRL-HeLa cells transfected with GFP-ER (green) exemplify the heterogeneity of ER expression levels. The cells show the RNA FISH signal associated with the array in the cell population (red signal) (I). The nuclear GFP-ER mean of fluorescence and RNA FISH array signal were plotted for vehicle- and E2-treated cells. Each symbol represents the measurements from a singe cell (II). C. To quantify FISH signal over 24 hours the total intensity of signal at the array (minus background signal) was determined by cumulative summation of 20 planes. Data represent the mean ±SEM of three different experiments graphed as fold induction over mean time-matched vehicle-treated control cells. D. This panel represents the earlier time points for E2 and EGF treatment. Fold activation between EGF and E2 at 30 minutes was significantly different, with a p value of 0.03.

    Techniques Used: Activity Assay, Expressing, Fluorescence In Situ Hybridization, Transfection, Fluorescence, Activation Assay

    57) Product Images from "Supported double membranes"

    Article Title: Supported double membranes

    Journal: Journal of structural biology

    doi: 10.1016/j.jsb.2009.02.008

    Membrane-tethered vesicles and supported double membrane. A: Biotinylated vesicles tethered by streptavidin to a biotin-PEG-doped supported bilayer. System components are not drawn to scale. B: Fluorescence micrograph of tethered vesicles, doped with Alexa647-syntaxin-1A. Scale bar: 10 µm. C: Supported double membrane. Tethered vesicles formed a second bilayer on top of the first; the inter-membrane space is bridged by the PEG-biotin-streptavidin-biotin-cap tethers. D: Fluorescence micrograph of a NBD-DOPE-doped supported double membrane. Scale bar: 10 µm.
    Figure Legend Snippet: Membrane-tethered vesicles and supported double membrane. A: Biotinylated vesicles tethered by streptavidin to a biotin-PEG-doped supported bilayer. System components are not drawn to scale. B: Fluorescence micrograph of tethered vesicles, doped with Alexa647-syntaxin-1A. Scale bar: 10 µm. C: Supported double membrane. Tethered vesicles formed a second bilayer on top of the first; the inter-membrane space is bridged by the PEG-biotin-streptavidin-biotin-cap tethers. D: Fluorescence micrograph of a NBD-DOPE-doped supported double membrane. Scale bar: 10 µm.

    Techniques Used: Fluorescence

    58) Product Images from "Stress release drives symmetry breaking for actin-based movement"

    Article Title: Stress release drives symmetry breaking for actin-based movement

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0502121102

    Effect of actin-regulating proteins and crosslinkers on gel growth and symmetry breaking. Beads with a radius of 3 μm were incubated in a medium containing 7.44 μM F-actin (10% labeled with Alexa Fluor 594) and, if not varied, 0.05 μM Arp2/3, 3 μM ADF/cofilin, and 0.2 μM gelsolin. ( a – c ) The effect of the concentrations of Arp2/3 (red), gelsolin (blue), ADF/cofilin (green), and profilin (black) on the initial growth velocity of the actin gel ( a ), the gel thickness h *( b ), and the average time 〈τ〉 at which symmetry breaking starts ( c ) is shown. Lines are shown as guides to assist viewing. ( d – f ) The effect of the concentrations of fascin (red), α-actinin (black), filamin (blue), and streptavidin (in the presence of 1.8 μM biotinylated actin) (green) on ( d ), h *( e ), and 〈τ〉 ( f ). Values were normalized with respect to the values in the absence of crosslinkers ( , ) to make trends more apparent. In all images, open symbols correspond with case I, filled symbols correspond with case II, and half-filled symbols are intermediate. Each point is an average of at least 10 measurements. The variance was 10–20% for h * and 20–40% for and 〈τ〉 in case I, and it could be up to 70% for 〈τ〉 in case II because of the nature of symmetry breaking under these conditions (see Discussion ). Plots of h as a function of time for several different concentrations of all proteins are shown in Figs. 7 and 8.
    Figure Legend Snippet: Effect of actin-regulating proteins and crosslinkers on gel growth and symmetry breaking. Beads with a radius of 3 μm were incubated in a medium containing 7.44 μM F-actin (10% labeled with Alexa Fluor 594) and, if not varied, 0.05 μM Arp2/3, 3 μM ADF/cofilin, and 0.2 μM gelsolin. ( a – c ) The effect of the concentrations of Arp2/3 (red), gelsolin (blue), ADF/cofilin (green), and profilin (black) on the initial growth velocity of the actin gel ( a ), the gel thickness h *( b ), and the average time 〈τ〉 at which symmetry breaking starts ( c ) is shown. Lines are shown as guides to assist viewing. ( d – f ) The effect of the concentrations of fascin (red), α-actinin (black), filamin (blue), and streptavidin (in the presence of 1.8 μM biotinylated actin) (green) on ( d ), h *( e ), and 〈τ〉 ( f ). Values were normalized with respect to the values in the absence of crosslinkers ( , ) to make trends more apparent. In all images, open symbols correspond with case I, filled symbols correspond with case II, and half-filled symbols are intermediate. Each point is an average of at least 10 measurements. The variance was 10–20% for h * and 20–40% for and 〈τ〉 in case I, and it could be up to 70% for 〈τ〉 in case II because of the nature of symmetry breaking under these conditions (see Discussion ). Plots of h as a function of time for several different concentrations of all proteins are shown in Figs. 7 and 8.

    Techniques Used: Incubation, Labeling

    59) Product Images from "Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR"

    Article Title: Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR

    Journal: eLife

    doi: 10.7554/eLife.50793

    The ∆∆ deletion does not affect the stability of the IRE1 LD dimer. ( A ) Bio-Layer Interferometry (BLI)-derived association (assoc.) and dissociation (dissoc.) traces of streptavidin sensors loaded with the indicated biotinylated ligands [wild-type (wt) or IRE1 LD ∆∆ ] and exposed sequentially to the indicated solutions of analyte. 2 µM BiP, 6.8 µM full-length ERdj4, or isolated J-domain and 2 mM ATP. A representative experiment of three independent repetitions is shown (processed as in Figure 5A ). ( B ) As in Figure 5A but comparing the monomeric J-IRE1 LD P108A and the disulphide-linked J-IRE1 LD Q105C SS proteins as ligands on the sensor. A representative experiment of three independent repetitions is shown. ( C ) Size-exclusion chromatography (SEC) elution profiles of TAMRA (TMR)-labelled wt and IRE1 LD ΔΔ proteins at the indicated concentrations. TMR fluorescence is plotted against elution time [note: fluorescent labelling was used to detect a signal at the low protein concentration (conc.) required to generate a pool of monomeric IRE1 LD ] (see: Figure 5—source data 2 ) ( D ) Plot of peak elution time points derived from ‘C’ against the protein concentration on a logarithmic scale for IRE1 LD and IRE1 LD ΔΔ . Curve fitting was performed in Prism GraphPad 7.0 using a sigmoidal function and calculated K 1/2 max values are displayed underneath the curves. Source data for Figure 5—figure supplement 1B. Source data for Figure 5—figure supplement 1C.
    Figure Legend Snippet: The ∆∆ deletion does not affect the stability of the IRE1 LD dimer. ( A ) Bio-Layer Interferometry (BLI)-derived association (assoc.) and dissociation (dissoc.) traces of streptavidin sensors loaded with the indicated biotinylated ligands [wild-type (wt) or IRE1 LD ∆∆ ] and exposed sequentially to the indicated solutions of analyte. 2 µM BiP, 6.8 µM full-length ERdj4, or isolated J-domain and 2 mM ATP. A representative experiment of three independent repetitions is shown (processed as in Figure 5A ). ( B ) As in Figure 5A but comparing the monomeric J-IRE1 LD P108A and the disulphide-linked J-IRE1 LD Q105C SS proteins as ligands on the sensor. A representative experiment of three independent repetitions is shown. ( C ) Size-exclusion chromatography (SEC) elution profiles of TAMRA (TMR)-labelled wt and IRE1 LD ΔΔ proteins at the indicated concentrations. TMR fluorescence is plotted against elution time [note: fluorescent labelling was used to detect a signal at the low protein concentration (conc.) required to generate a pool of monomeric IRE1 LD ] (see: Figure 5—source data 2 ) ( D ) Plot of peak elution time points derived from ‘C’ against the protein concentration on a logarithmic scale for IRE1 LD and IRE1 LD ΔΔ . Curve fitting was performed in Prism GraphPad 7.0 using a sigmoidal function and calculated K 1/2 max values are displayed underneath the curves. Source data for Figure 5—figure supplement 1B. Source data for Figure 5—figure supplement 1C.

    Techniques Used: Derivative Assay, Isolation, Size-exclusion Chromatography, Fluorescence, Protein Concentration

    60) Product Images from "Monitoring Phosphatidic Acid Formation in Intact Phosphatidylcholine Bilayers upon Phospholipase D Catalysis"

    Article Title: Monitoring Phosphatidic Acid Formation in Intact Phosphatidylcholine Bilayers upon Phospholipase D Catalysis

    Journal: Analytical Chemistry

    doi: 10.1021/ac403580r

    Electrophoretic-electroosmotic focusing (EEF) of Texas Red-DHPE and streptavidin biomarkers on SLBs after PLD-catalyzed POPA formation with (A) 0, (B) 2.5, (C) 5, and (D) 10 mM Ca 2+ concentrations. The scale bars are 100 μm.
    Figure Legend Snippet: Electrophoretic-electroosmotic focusing (EEF) of Texas Red-DHPE and streptavidin biomarkers on SLBs after PLD-catalyzed POPA formation with (A) 0, (B) 2.5, (C) 5, and (D) 10 mM Ca 2+ concentrations. The scale bars are 100 μm.

    Techniques Used:

    Line-scan fluorescence profiles of the streptavidin focusing positions on SLBs after the PLD-catalyzed reactions in Figure 4 . All SLBs were incubated with PLD and Ca 2+ for 10 min.
    Figure Legend Snippet: Line-scan fluorescence profiles of the streptavidin focusing positions on SLBs after the PLD-catalyzed reactions in Figure 4 . All SLBs were incubated with PLD and Ca 2+ for 10 min.

    Techniques Used: Fluorescence, Incubation

    Effect of POPA concentration on the focusing position of streptavidin. (A) The expected focusing position calculated as a function of POPA concentration in the SLB. 23 (B) Focusing of Texas Red-DHPE and streptavidin with 0 and 10 mol % PA percentages. The ■ along the blue line in (A) represent the actual focusing positions found from the corresponding maxima in fluorescent intensity from the streptavidin in (B).
    Figure Legend Snippet: Effect of POPA concentration on the focusing position of streptavidin. (A) The expected focusing position calculated as a function of POPA concentration in the SLB. 23 (B) Focusing of Texas Red-DHPE and streptavidin with 0 and 10 mol % PA percentages. The ■ along the blue line in (A) represent the actual focusing positions found from the corresponding maxima in fluorescent intensity from the streptavidin in (B).

    Techniques Used: Concentration Assay

    61) Product Images from "Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells"

    Article Title: Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki968

    SAABS assay. ( A ) SAABS procedure. A random 8mer oligodeoxynucleotide library (ROL) flanked by two PCR tags is first incubated with biotinylated mRNA. The mRNA bound ODN is then separated from free ODN by binding the biotinylated mRNA to a streptavidin coated Dynabead, which is then separated from the unbound sequence by a magnetic field. The bound sequence is then PCR amplified with S1 and CS2, restricted with NlaIII, concatenated by ligation, cloned in pZErO-1 and sequenced. ( B ) Frequency distribution of the antisense binding sites on the unr mRNA obtained from the SAABS assay. The 8mer sequences were retrieved from the sequenced clones and aligned with the mRNA sequence. Some of the sites identified correspond to sites found by the RT-ROL assay (13 and 46), whereas others were uniquely detected by the SAABS assay and denoted with an S prefix (S1, S3, S5 and S7).
    Figure Legend Snippet: SAABS assay. ( A ) SAABS procedure. A random 8mer oligodeoxynucleotide library (ROL) flanked by two PCR tags is first incubated with biotinylated mRNA. The mRNA bound ODN is then separated from free ODN by binding the biotinylated mRNA to a streptavidin coated Dynabead, which is then separated from the unbound sequence by a magnetic field. The bound sequence is then PCR amplified with S1 and CS2, restricted with NlaIII, concatenated by ligation, cloned in pZErO-1 and sequenced. ( B ) Frequency distribution of the antisense binding sites on the unr mRNA obtained from the SAABS assay. The 8mer sequences were retrieved from the sequenced clones and aligned with the mRNA sequence. Some of the sites identified correspond to sites found by the RT-ROL assay (13 and 46), whereas others were uniquely detected by the SAABS assay and denoted with an S prefix (S1, S3, S5 and S7).

    Techniques Used: Polymerase Chain Reaction, Incubation, Binding Assay, Sequencing, Amplification, Ligation, Clone Assay

    Dynabead-based dot blot assay to determine relative binding affinity of ODNs. ( A ) Determining the loading capacity of the streptavidin coated Dynabead by titrating 20 µl of bead solution in 40 µl total volume of 0.5 M NaCl with biotinylated radiolabeled unr mRNA. ( B ) Determining the µl of Dynabead bound RNA needed to completely bind 1 pmol of ODN5 in a total volume of 40 µl. ( C and D ) Solutions of RNA were incubated with 1 pmol of ODN [1–54 from RT-ROL assay and 57–68 (S1-S12) from the SAABS assay] and then incubated with 10 µl of Dynabeads and spotted on Nylon membrane. (C) is a photograph of blot showing equal loading of beads. (D) is a radiogram showing relative amounts of retained ODN. ODNs corresponding to circled spots were further studied by quantitative methods.
    Figure Legend Snippet: Dynabead-based dot blot assay to determine relative binding affinity of ODNs. ( A ) Determining the loading capacity of the streptavidin coated Dynabead by titrating 20 µl of bead solution in 40 µl total volume of 0.5 M NaCl with biotinylated radiolabeled unr mRNA. ( B ) Determining the µl of Dynabead bound RNA needed to completely bind 1 pmol of ODN5 in a total volume of 40 µl. ( C and D ) Solutions of RNA were incubated with 1 pmol of ODN [1–54 from RT-ROL assay and 57–68 (S1-S12) from the SAABS assay] and then incubated with 10 µl of Dynabeads and spotted on Nylon membrane. (C) is a photograph of blot showing equal loading of beads. (D) is a radiogram showing relative amounts of retained ODN. ODNs corresponding to circled spots were further studied by quantitative methods.

    Techniques Used: Dot Blot, Binding Assay, Incubation

    62) Product Images from "A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes *"

    Article Title: A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.444414

    Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or neutravidin beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.
    Figure Legend Snippet: Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or neutravidin beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.

    Techniques Used: In Vitro, Selection, Binding Assay, Concentration Assay, Polymerase Chain Reaction

    63) Product Images from "Cinnamate-based DNA photolithography"

    Article Title: Cinnamate-based DNA photolithography

    Journal: Nature materials

    doi: 10.1038/nmat3645

    Schematic protocol for DNA Photo-Lithography with cinnamate-based DNA strands a) Schematic representations of the DNA constructs and the position of cinnamate cross-linkers. The sequences are: Surface Strand : 5′-RSS-50basesBackbone-TTGAGAAATGC-cinnamate-CGTAAAGAGTT-3′; Linker Strand: 5′-CATCTTCATCCAACTCTTTACG-cinnamate-GCATTTCTCAA-3′; Particle Strand: 5′-GGATGAAGATG-50basesBackbone-BiotinTEG-3′; Linker Strand 2: 5′-BiotinTEG- AACTCTTTACG-cinnamate-GCATTTCTCAA-3′. b) Procedures to fabricate the multi-functionalized DNA surface. Steps 1 and 2: hybridization of linker-strands (LS) to surface strands (SS) (purple section of DNA strand) by cooling them from 55 °C to 25 °C over a period of 30 minutes; Step 3: permanently UV cross-link SS and LS in selected area (purple section with solid black dot); Step 4: heating the sample to 55°C to de-hybridize unlinked strands and washing of the sample. The functionalized DNA surface is then ready for use, e.g. by reversibly binding colloids (step 5) to the patterned regions, or by use of fluorescently labeled complementary DNA strand (step 6). To add another function to the surface, linker strand 2 (LS2) needs to be hybridized to the SS and then UV cross-linked in the desired region (step 7). The multi-functionalized surface is the ready for use (after being heated to 55°C and washed (step 8)). We can visualize the patterns by red streptavidin binding to biotin in the LS2 patterned region, and green fluorescently labeled PS hybridizing to LS patterned region (step 9).
    Figure Legend Snippet: Schematic protocol for DNA Photo-Lithography with cinnamate-based DNA strands a) Schematic representations of the DNA constructs and the position of cinnamate cross-linkers. The sequences are: Surface Strand : 5′-RSS-50basesBackbone-TTGAGAAATGC-cinnamate-CGTAAAGAGTT-3′; Linker Strand: 5′-CATCTTCATCCAACTCTTTACG-cinnamate-GCATTTCTCAA-3′; Particle Strand: 5′-GGATGAAGATG-50basesBackbone-BiotinTEG-3′; Linker Strand 2: 5′-BiotinTEG- AACTCTTTACG-cinnamate-GCATTTCTCAA-3′. b) Procedures to fabricate the multi-functionalized DNA surface. Steps 1 and 2: hybridization of linker-strands (LS) to surface strands (SS) (purple section of DNA strand) by cooling them from 55 °C to 25 °C over a period of 30 minutes; Step 3: permanently UV cross-link SS and LS in selected area (purple section with solid black dot); Step 4: heating the sample to 55°C to de-hybridize unlinked strands and washing of the sample. The functionalized DNA surface is then ready for use, e.g. by reversibly binding colloids (step 5) to the patterned regions, or by use of fluorescently labeled complementary DNA strand (step 6). To add another function to the surface, linker strand 2 (LS2) needs to be hybridized to the SS and then UV cross-linked in the desired region (step 7). The multi-functionalized surface is the ready for use (after being heated to 55°C and washed (step 8)). We can visualize the patterns by red streptavidin binding to biotin in the LS2 patterned region, and green fluorescently labeled PS hybridizing to LS patterned region (step 9).

    Techniques Used: Construct, Hybridization, Binding Assay, Labeling

    DNA photo-lithographic pattern on surface (a) and (b): right: exposed patterns with different feature sizes (∼14μm in (a) and 4μm and 1.5μm in (b)); left: colloid patterns (inverted so unstuck particles have escaped); middle: Green fluorescently labeled conjugate images. (c) Multi-functionalized patterns of ‘nature materials’ (abbreviated as ‘n m’) in New Times Roman font. Left: Green fluorescently labeled DNA hybridizes to LS patterned in the region ‘materials,’ and red streptavidin binds to LS2 patterned biotin in the region ‘nature’. Right: similar patterning of larger ‘n’ and ‘m’ letters. Separate confocal filtering channels are shown as smaller figures.
    Figure Legend Snippet: DNA photo-lithographic pattern on surface (a) and (b): right: exposed patterns with different feature sizes (∼14μm in (a) and 4μm and 1.5μm in (b)); left: colloid patterns (inverted so unstuck particles have escaped); middle: Green fluorescently labeled conjugate images. (c) Multi-functionalized patterns of ‘nature materials’ (abbreviated as ‘n m’) in New Times Roman font. Left: Green fluorescently labeled DNA hybridizes to LS patterned in the region ‘materials,’ and red streptavidin binds to LS2 patterned biotin in the region ‘nature’. Right: similar patterning of larger ‘n’ and ‘m’ letters. Separate confocal filtering channels are shown as smaller figures.

    Techniques Used: Labeling

    64) Product Images from "In Vitro Selection of Proteins with Desired Characteristics Using mRNA-display"

    Article Title: In Vitro Selection of Proteins with Desired Characteristics Using mRNA-display

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2012.11.004

    Identification of two granzyme B substrates by using a selection procedure that allows the identification of family member specific substrates. Prior to selection, the mRNA-displayed proteome library is immobilized on a streptavidin-agarose matrix and cleaved with other caspase members to remove the overlapping substrates. After stringent washing, the pre-cleared proteome library was used to enrich for the family member-specific substrates that are cleaved by the protease of interest, in this case, granzyme B (GB). The numbers 1 to 10 indicate the caspase used in the proteolytic assay. Caspase-10a and 10b refer the two isoforms of caspase-10. The 35 S-labeled full-length proteins are shown below (arrow).
    Figure Legend Snippet: Identification of two granzyme B substrates by using a selection procedure that allows the identification of family member specific substrates. Prior to selection, the mRNA-displayed proteome library is immobilized on a streptavidin-agarose matrix and cleaved with other caspase members to remove the overlapping substrates. After stringent washing, the pre-cleared proteome library was used to enrich for the family member-specific substrates that are cleaved by the protease of interest, in this case, granzyme B (GB). The numbers 1 to 10 indicate the caspase used in the proteolytic assay. Caspase-10a and 10b refer the two isoforms of caspase-10. The 35 S-labeled full-length proteins are shown below (arrow).

    Techniques Used: Selection, Labeling

    Sample selection types. Schematic representation of the selection procedure for the natural substrates of a protease of interest from an mRNA-displayed proteome library (Option A). Schematic representation of the selection procedure for the binding partners of a target of interest from an mRNA-displayed proteome library (Option B). mRNA, blue; DNA, black; protein, orange; puromycin, pink circle labeled with a P; biotin, black circle labeled with a B; streptavidin, black circle labeled with an S; agarose beads, yellow circle labeled as agarose; protease, black scissors; target protein, red.
    Figure Legend Snippet: Sample selection types. Schematic representation of the selection procedure for the natural substrates of a protease of interest from an mRNA-displayed proteome library (Option A). Schematic representation of the selection procedure for the binding partners of a target of interest from an mRNA-displayed proteome library (Option B). mRNA, blue; DNA, black; protein, orange; puromycin, pink circle labeled with a P; biotin, black circle labeled with a B; streptavidin, black circle labeled with an S; agarose beads, yellow circle labeled as agarose; protease, black scissors; target protein, red.

    Techniques Used: Selection, Binding Assay, Labeling

    65) Product Images from "The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells"

    Article Title: The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

    Journal: The Journal of Cell Biology

    doi:

    Effect of A19 oligonucleotide on the polarity of sucrase isomaltase and alkaline phosphatase in CACO-2 cells. Confluent monolayers were grown on polycarbonate filters in the presence of random (lanes A and B ) or A19 (lanes C and D ) oligonucleotides as described above. The cells were biotinylated from the apical (lanes A and C ) or basolateral sides (lanes B and D ) and consecutively extracted in Triton X-114 at 4°C and with Triton X-100 at 37°C. The Triton X-114 detergent phase and the Triton X-100 supernatant were pooled and affinity purified with streptavidin–agarose. The eluates or the affinity purification step were then analyzed by immunoblot with antibodies against either sucrase isomaltase or alkaline phosphatase and a chemiluminescence detection system. The average OD measures obtained from unfiltered digitized images after subracting background from the average pixel value over the band of each lane were as follows (scale 0-255): ( Sucrase A ) 58; ( B ) 14; ( C ) 55; ( D ) 29; ( Alk. Phosphatase, A ) 74; ( B ) 1; ( C ) 30; ( D ) 16. Weighted OD measures (average pixel value × number of pixels) were as follows (in thousands): ( Sucrase, A ) 141; ( B ) 10; ( C ) 169; ( D ) 35; ( Alk. Phosphatase, A ) 45; ( B ) 0.6; ( C ) 36; ( D ) 5.
    Figure Legend Snippet: Effect of A19 oligonucleotide on the polarity of sucrase isomaltase and alkaline phosphatase in CACO-2 cells. Confluent monolayers were grown on polycarbonate filters in the presence of random (lanes A and B ) or A19 (lanes C and D ) oligonucleotides as described above. The cells were biotinylated from the apical (lanes A and C ) or basolateral sides (lanes B and D ) and consecutively extracted in Triton X-114 at 4°C and with Triton X-100 at 37°C. The Triton X-114 detergent phase and the Triton X-100 supernatant were pooled and affinity purified with streptavidin–agarose. The eluates or the affinity purification step were then analyzed by immunoblot with antibodies against either sucrase isomaltase or alkaline phosphatase and a chemiluminescence detection system. The average OD measures obtained from unfiltered digitized images after subracting background from the average pixel value over the band of each lane were as follows (scale 0-255): ( Sucrase A ) 58; ( B ) 14; ( C ) 55; ( D ) 29; ( Alk. Phosphatase, A ) 74; ( B ) 1; ( C ) 30; ( D ) 16. Weighted OD measures (average pixel value × number of pixels) were as follows (in thousands): ( Sucrase, A ) 141; ( B ) 10; ( C ) 169; ( D ) 35; ( Alk. Phosphatase, A ) 45; ( B ) 0.6; ( C ) 36; ( D ) 5.

    Techniques Used: Affinity Purification

    Effect of antisense A19 oligonucleotide on the polarity of plasma membrane proteins in CACO-2 cells C2BBe1. The cells were continuously grown in random (lanes A , B , E , F , I , and J ; control) or antisense A19 (lanes C , D , G , H , K , and L ) oligonucleotides. For these experiments, the cells were plated on 24-mm Transwell TM filters and cultured at confluency for 9 d. The monolayers were biotinylated from either the apical side (lanes A , C , E , G , I , K ; Ap ) or from the basolateral side (lanes B , D , F , H , J , L ; Bl ). Then, the filters were extracted in ice-cold PBS-EDTA supplemented with 2% Triton X-114. The supernatant of this extraction was warmed to 30°C for 3 min, and the detergent phase was acetone precipitated and run in SDS-PAGE (lanes I–L ; TX-114 ). The pellets from the TX-114 extraction were then resuspended in PBS-EDTA, 1% Triton X-100 by sonication, and warmed up to 37°C for 15 min. The supernatants (lanes E–H ; TX-100 ) and pellets (lanes A–D ; Pellet ) of this second extraction were also run in SDS-PAGE. In all cases the total amount of protein was measured to ensure that all lanes for a given extraction procedure were seeded with the same amounts of cellular material. All the lanes were blotted onto nitrocellulose sheets and probed with streptavidin–peroxidase and a chemiluminescence reaction. The small arrows between K and L point at apical bands now appearing also in the basolateral labeled set of proteins. The arrowheads indicate the position of molecular weight standards: (lanes A–D ) 193, 112, 86, 70, 57, and 36 kD; (lanes E–L ) 205, 116, 66, 45, and 29 kD. All the blots are from the same experiment, although, for technical reasons, they were run in separate gels with two different sets of molecular weight standards. Biotinylation control: CACO-2 C2BBe monolayers were grown on filters, incubated in A19, and biotinylated as described above. The cells were extensively washed, fixed in PFA, and processed with fluorescein-coupled streptavidin from both sides of the filter. Optical confocal sections were taken at the transnuclear plane ( a ) or at the apical membrane plane ( b ). Bars, 10 μm.
    Figure Legend Snippet: Effect of antisense A19 oligonucleotide on the polarity of plasma membrane proteins in CACO-2 cells C2BBe1. The cells were continuously grown in random (lanes A , B , E , F , I , and J ; control) or antisense A19 (lanes C , D , G , H , K , and L ) oligonucleotides. For these experiments, the cells were plated on 24-mm Transwell TM filters and cultured at confluency for 9 d. The monolayers were biotinylated from either the apical side (lanes A , C , E , G , I , K ; Ap ) or from the basolateral side (lanes B , D , F , H , J , L ; Bl ). Then, the filters were extracted in ice-cold PBS-EDTA supplemented with 2% Triton X-114. The supernatant of this extraction was warmed to 30°C for 3 min, and the detergent phase was acetone precipitated and run in SDS-PAGE (lanes I–L ; TX-114 ). The pellets from the TX-114 extraction were then resuspended in PBS-EDTA, 1% Triton X-100 by sonication, and warmed up to 37°C for 15 min. The supernatants (lanes E–H ; TX-100 ) and pellets (lanes A–D ; Pellet ) of this second extraction were also run in SDS-PAGE. In all cases the total amount of protein was measured to ensure that all lanes for a given extraction procedure were seeded with the same amounts of cellular material. All the lanes were blotted onto nitrocellulose sheets and probed with streptavidin–peroxidase and a chemiluminescence reaction. The small arrows between K and L point at apical bands now appearing also in the basolateral labeled set of proteins. The arrowheads indicate the position of molecular weight standards: (lanes A–D ) 193, 112, 86, 70, 57, and 36 kD; (lanes E–L ) 205, 116, 66, 45, and 29 kD. All the blots are from the same experiment, although, for technical reasons, they were run in separate gels with two different sets of molecular weight standards. Biotinylation control: CACO-2 C2BBe monolayers were grown on filters, incubated in A19, and biotinylated as described above. The cells were extensively washed, fixed in PFA, and processed with fluorescein-coupled streptavidin from both sides of the filter. Optical confocal sections were taken at the transnuclear plane ( a ) or at the apical membrane plane ( b ). Bars, 10 μm.

    Techniques Used: Cell Culture, SDS Page, Sonication, Labeling, Molecular Weight, Incubation

    Uptake and effect of antisense (A19) oligonucleotide in MCF-10A and CACO-2 cells. MCF-10A ( a and b ) and CACO-2 cells ( c–j ) were cultured on glass coverslips. The cells were continuously grown in either random ( a , c , e , g , i ) or A19 ( b , d , f , h , j ) oligonucleotides. In some cases, the cells were fixed (PFA), permeabilized, and processed with a biotinylated sense oligonucleotide (complementary of A19 , a–d ; or complementary of random , e and f ) followed by Texas red–conjugated streptavidin. Other monolayers were processed for indirect immunofluorescence with anti-CK19 mAb (RCK108; g–j ). Some samples were observed under laser confocal microscopy as a series of confocal optical sections in the z axis (perpendicular to the plane of the monolayer), and then the transmonolayer section image was obtained as a three-dimensional reconstruction of 4 voxel thick volumes ( g and h ). Black arrowheads point to the apical CK19 network, and white arrowheads show the position of the basal domain. Other samples are shown desuper under standard epifluorescence microscopy ( i and j ). Bars: ( a–f , i , j ) 10 μm; ( g and h ) 20 μm.
    Figure Legend Snippet: Uptake and effect of antisense (A19) oligonucleotide in MCF-10A and CACO-2 cells. MCF-10A ( a and b ) and CACO-2 cells ( c–j ) were cultured on glass coverslips. The cells were continuously grown in either random ( a , c , e , g , i ) or A19 ( b , d , f , h , j ) oligonucleotides. In some cases, the cells were fixed (PFA), permeabilized, and processed with a biotinylated sense oligonucleotide (complementary of A19 , a–d ; or complementary of random , e and f ) followed by Texas red–conjugated streptavidin. Other monolayers were processed for indirect immunofluorescence with anti-CK19 mAb (RCK108; g–j ). Some samples were observed under laser confocal microscopy as a series of confocal optical sections in the z axis (perpendicular to the plane of the monolayer), and then the transmonolayer section image was obtained as a three-dimensional reconstruction of 4 voxel thick volumes ( g and h ). Black arrowheads point to the apical CK19 network, and white arrowheads show the position of the basal domain. Other samples are shown desuper under standard epifluorescence microscopy ( i and j ). Bars: ( a–f , i , j ) 10 μm; ( g and h ) 20 μm.

    Techniques Used: Cell Culture, Immunofluorescence, Confocal Microscopy, Epifluorescence Microscopy

    66) Product Images from "Optimized Light-Directed Synthesis of Aptamer Microarrays"

    Article Title: Optimized Light-Directed Synthesis of Aptamer Microarrays

    Journal: Analytical Chemistry

    doi: 10.1021/ac400746j

    Fluorescent images of sections of microarrays synthesized on different substrates. (A) Corning UltraGAPS, (B) Schott Epoxy ring-opened, (C) Schott A+, (D) Schott Glass D hydroxyl-functionalized with N -(3-triethoxysilylpropyl)-4-hydroxybutryramide. Green features are hybridization signals from Cy3-labeled sequences. Red features are from Cy5-labeled biotin binding to the streptavidin–aptamer pairs. A scheme identifying the sequences corresponding to the spots can be found in the Supporting Information . The arrays were synthesized with light exposures of 11 J/cm 2 and with 32 × 32 μm features (4 DMD mirrors) separated by gaps of 48 μm. All images were acquired with the same scanner settings.
    Figure Legend Snippet: Fluorescent images of sections of microarrays synthesized on different substrates. (A) Corning UltraGAPS, (B) Schott Epoxy ring-opened, (C) Schott A+, (D) Schott Glass D hydroxyl-functionalized with N -(3-triethoxysilylpropyl)-4-hydroxybutryramide. Green features are hybridization signals from Cy3-labeled sequences. Red features are from Cy5-labeled biotin binding to the streptavidin–aptamer pairs. A scheme identifying the sequences corresponding to the spots can be found in the Supporting Information . The arrays were synthesized with light exposures of 11 J/cm 2 and with 32 × 32 μm features (4 DMD mirrors) separated by gaps of 48 μm. All images were acquired with the same scanner settings.

    Techniques Used: Synthesized, Hybridization, Labeling, Binding Assay

    Aptamer binding and hybridization signal comparison between microarrays synthesized with four different surface chemistries (left to right): Corning UltraGAPS/amino-modified; Schott E/epoxy-modified, ring-opened; Schott A+/amino-modified; and in-house hydroxyl-functionalized. Top: St-2-1 aptamer–streptavidin binding signal. Bottom: hybridization signal for three probes hybridized with 1 and 100 pM complementary sequences. Error bars are the standard deviation among replicates.
    Figure Legend Snippet: Aptamer binding and hybridization signal comparison between microarrays synthesized with four different surface chemistries (left to right): Corning UltraGAPS/amino-modified; Schott E/epoxy-modified, ring-opened; Schott A+/amino-modified; and in-house hydroxyl-functionalized. Top: St-2-1 aptamer–streptavidin binding signal. Bottom: hybridization signal for three probes hybridized with 1 and 100 pM complementary sequences. Error bars are the standard deviation among replicates.

    Techniques Used: Binding Assay, Hybridization, Synthesized, Modification, Standard Deviation

    Effect of mutations in the 29-mer St-2-1 aptamer sequence on the binding affinity. Binding was tested using an on-array streptavidin binding assay. Black bar, St-2-1; blue bars, mutation in the terminal stem; green bars, mutation in the bulges; red bars, mutation in the sequences between the bulges. Error bars are based on 12 replicates on the array. Signal negative control St-2-1_rev =28.4.
    Figure Legend Snippet: Effect of mutations in the 29-mer St-2-1 aptamer sequence on the binding affinity. Binding was tested using an on-array streptavidin binding assay. Black bar, St-2-1; blue bars, mutation in the terminal stem; green bars, mutation in the bulges; red bars, mutation in the sequences between the bulges. Error bars are based on 12 replicates on the array. Signal negative control St-2-1_rev =28.4.

    Techniques Used: Sequencing, Binding Assay, Mutagenesis, Negative Control

    Effect of base pair replacements in the double-stranded parts of St-2-1. Binding was tested using an on-array streptavidin binding assay. Black bar, St-2-1; blue bars, mutation in the terminal stem; red bars, mutation in the sequence between the bulges. Error bars are based on 12 replicates on the array. Secondary structure of the variants A 12 T 23 , G 12 C 23 , and C 12 G 23 . The structural variant C 12 G 23 correlates with reduced streptavidin binding on-array.
    Figure Legend Snippet: Effect of base pair replacements in the double-stranded parts of St-2-1. Binding was tested using an on-array streptavidin binding assay. Black bar, St-2-1; blue bars, mutation in the terminal stem; red bars, mutation in the sequence between the bulges. Error bars are based on 12 replicates on the array. Secondary structure of the variants A 12 T 23 , G 12 C 23 , and C 12 G 23 . The structural variant C 12 G 23 correlates with reduced streptavidin binding on-array.

    Techniques Used: Binding Assay, Mutagenesis, Sequencing, Variant Assay

    Normalized streptavidin binding signal for a St-2-1 aptamer sequence synthesized with a photodeprotection light exposure gradient between 0.2 and 18 J/cm 2 . The black squares represent data from a microarray synthesized with a maximum surface density of oligonucleotides. The red circles are from an equivalent experiment, but with a microarray with an oligonucleotide surface density reduced by 50%.
    Figure Legend Snippet: Normalized streptavidin binding signal for a St-2-1 aptamer sequence synthesized with a photodeprotection light exposure gradient between 0.2 and 18 J/cm 2 . The black squares represent data from a microarray synthesized with a maximum surface density of oligonucleotides. The red circles are from an equivalent experiment, but with a microarray with an oligonucleotide surface density reduced by 50%.

    Techniques Used: Binding Assay, Sequencing, Synthesized, Microarray

    67) Product Images from "Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane"

    Article Title: Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200605142

    P. aeruginosa induces the relocalization of proteins from the BL to the AP surface by transcytosis. (a, left) The BL surface of confluent MDCK cells was biotinylated for 30 min at 4°C. Cells were infected with P. aeruginosa expressing GFP for 30 min at 37°C, fixed, and the AP and BL surfaces were probed with fluorescent streptavidin (red). Biotinylated BL proteins are only detectable on the BL surface and at the sites of bacterial attachment on the AP surface. (a, right) The relocalization of BL proteins is reduced by LY. Cells were preincubated with 100 μM LY for 1 h. The drug was also present during biotinylation and infection. (b) The diffusion from the AP to the BL surface of the small molecule FITC-inulin was measured in uninfected cells, cells infected with P. aeruginosa for 30 min, or after exposure to 10 mM EDTA for 10 min. AP addition of P. aeruginosa decreased the monolayer permeability. Error bars represent SD. (c) XZ section of MDCK cells infected with GFP– P. aeruginosa and stained with an antibody against an extracellular domain of the BL protein β1-integrin (red). The cells were then fixed and stained with DAPI (blue) to visualize the nuclei. The addition of bacteria did not permit diffusion of the antibody to the BL surface, suggesting that the tight junctions were not disrupted upon bacterial binding. (d) BL surface biotinylation of MDCK T23 cells previously infected with recombinant adenovirus encoding HA-tagged DN dynamin II was performed as described in panel a. DN dynamin II (blue) is expressed both in AP and BL membranes. The left panel shows a typical example of the relocalization of biotinylated BL proteins to the site of AP bacterial attachment to a nontransfected cell. The right panel shows that in cells expressing DN dynamin II, the AP accumulation of biotinylated proteins was inhibited. Bars, 10 μm.
    Figure Legend Snippet: P. aeruginosa induces the relocalization of proteins from the BL to the AP surface by transcytosis. (a, left) The BL surface of confluent MDCK cells was biotinylated for 30 min at 4°C. Cells were infected with P. aeruginosa expressing GFP for 30 min at 37°C, fixed, and the AP and BL surfaces were probed with fluorescent streptavidin (red). Biotinylated BL proteins are only detectable on the BL surface and at the sites of bacterial attachment on the AP surface. (a, right) The relocalization of BL proteins is reduced by LY. Cells were preincubated with 100 μM LY for 1 h. The drug was also present during biotinylation and infection. (b) The diffusion from the AP to the BL surface of the small molecule FITC-inulin was measured in uninfected cells, cells infected with P. aeruginosa for 30 min, or after exposure to 10 mM EDTA for 10 min. AP addition of P. aeruginosa decreased the monolayer permeability. Error bars represent SD. (c) XZ section of MDCK cells infected with GFP– P. aeruginosa and stained with an antibody against an extracellular domain of the BL protein β1-integrin (red). The cells were then fixed and stained with DAPI (blue) to visualize the nuclei. The addition of bacteria did not permit diffusion of the antibody to the BL surface, suggesting that the tight junctions were not disrupted upon bacterial binding. (d) BL surface biotinylation of MDCK T23 cells previously infected with recombinant adenovirus encoding HA-tagged DN dynamin II was performed as described in panel a. DN dynamin II (blue) is expressed both in AP and BL membranes. The left panel shows a typical example of the relocalization of biotinylated BL proteins to the site of AP bacterial attachment to a nontransfected cell. The right panel shows that in cells expressing DN dynamin II, the AP accumulation of biotinylated proteins was inhibited. Bars, 10 μm.

    Techniques Used: Infection, Expressing, Diffusion-based Assay, Permeability, Staining, Binding Assay, Recombinant

    68) Product Images from "Recognition of host Clr-b by the inhibitory NKR-P1B receptor provides a basis for missing-self recognition"

    Article Title: Recognition of host Clr-b by the inhibitory NKR-P1B receptor provides a basis for missing-self recognition

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06989-2

    Energetic basis of the NKR-P1B:Clr-b interaction. a BWZ cells (top) or HEK293T cells (bottom) were transduced or transfected, respectively, with empty vector (dashed line) or Clr-b-expressing vector (shaded gray), and stained using anti-Clr-b antibody (left) or NKR-P1B tetramers (right) and analyzed by flow cytometry. b HEK293T cells were transfected with vector expressing Clr-b (pIRES2-EGFP), and 48 h later were stained with NKR-P1B mutant tetramers. The GFP expression measures transfection efficiency and PE measures binding by PE-tetramers. Gates were set up using untransfected cells (HEK293T) and cells transfected with empty pIRES2-EGFP (Vector) that were stained with NKR-P1B tetramer (WT) or Streptavidin-PE (SA-PE). Labels on the top left correspond to point mutation on the NKR-P1B molecule, whereas italicized numbers correspond to mean fluorescence intensity of NKR-P1B. c Cells were transfected with constructs expressing NKR-P1B mutants, and 48 h later were used as stimulators to BWZ.CD3ζ/Clr-b reporters. Co-cultures were setup using a 1:1 stimulators: reporters ratio, and the next day were assayed for production of β-galactosidase using colorimetric assay. d , e NKR-P1B dimer mutants were transfected into HEK293T cells and used in BWZ assays using 3-fold dilutions of stimulators against BWZ.CD3ζ/Clr-b reporters (left) or BWZ.CD3ζ/m12 Smith reporters (right). Significant differences are shown between the WT and mutant allele for each graph. Data were analyzed using ( c ) one-way ANOVA [F(15,48) = 51.88, p
    Figure Legend Snippet: Energetic basis of the NKR-P1B:Clr-b interaction. a BWZ cells (top) or HEK293T cells (bottom) were transduced or transfected, respectively, with empty vector (dashed line) or Clr-b-expressing vector (shaded gray), and stained using anti-Clr-b antibody (left) or NKR-P1B tetramers (right) and analyzed by flow cytometry. b HEK293T cells were transfected with vector expressing Clr-b (pIRES2-EGFP), and 48 h later were stained with NKR-P1B mutant tetramers. The GFP expression measures transfection efficiency and PE measures binding by PE-tetramers. Gates were set up using untransfected cells (HEK293T) and cells transfected with empty pIRES2-EGFP (Vector) that were stained with NKR-P1B tetramer (WT) or Streptavidin-PE (SA-PE). Labels on the top left correspond to point mutation on the NKR-P1B molecule, whereas italicized numbers correspond to mean fluorescence intensity of NKR-P1B. c Cells were transfected with constructs expressing NKR-P1B mutants, and 48 h later were used as stimulators to BWZ.CD3ζ/Clr-b reporters. Co-cultures were setup using a 1:1 stimulators: reporters ratio, and the next day were assayed for production of β-galactosidase using colorimetric assay. d , e NKR-P1B dimer mutants were transfected into HEK293T cells and used in BWZ assays using 3-fold dilutions of stimulators against BWZ.CD3ζ/Clr-b reporters (left) or BWZ.CD3ζ/m12 Smith reporters (right). Significant differences are shown between the WT and mutant allele for each graph. Data were analyzed using ( c ) one-way ANOVA [F(15,48) = 51.88, p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Cytometry, Mutagenesis, Binding Assay, Fluorescence, Construct, Colorimetric Assay

    69) Product Images from "Exclusive formation of monovalent quantum dot imaging probes by steric exclusion"

    Article Title: Exclusive formation of monovalent quantum dot imaging probes by steric exclusion

    Journal: Nature methods

    doi: 10.1038/nmeth.2682

    ptDNA-wrapped QDs are monovalent and do not oligomerize protein targets on supported lipid bilayers (a) Representative TEM images of (left) commercial Streptavidin QDots incubated with gold nanoparticles bearing a biotinylated DNA sequence and (right) mQDs hybridized with gold nanoparticles bearing a complementary ssDNA sequence. ( b ) Statistical analyses of QD valencies from TEM images reveals the monovalent character of mQDs (Orange bars, n =545) compared with Streptavidin QDots (purple bars, n =188). ( c ) Distribution of diffusion constants of streptavidin-linked SNAP proteins on supported lipid bilayers (thin purple line, n =756, 0.56 μm 2 /s mean) shows a decrease when the concentration of protein is increased 100 fold (thick purple line, n =189, 0.35 μm 2 /s mean). Distribution of diffusion constants of mQD-linked SNAP proteins (thin orange line, n =490, 0.89 μm 2 /s mean) is not altered when protein concentration is increased 100-fold (thick orange line n =790 0.86 μm 2 /s mean). SNAP protein diffusion rates measured with mQDs are nearly identical to diffusion rates measure with small organic dyes such as Atto488 (dotted black line, n =245, 0.89 μm 2 /s mean). Scale bar = 25 nm.
    Figure Legend Snippet: ptDNA-wrapped QDs are monovalent and do not oligomerize protein targets on supported lipid bilayers (a) Representative TEM images of (left) commercial Streptavidin QDots incubated with gold nanoparticles bearing a biotinylated DNA sequence and (right) mQDs hybridized with gold nanoparticles bearing a complementary ssDNA sequence. ( b ) Statistical analyses of QD valencies from TEM images reveals the monovalent character of mQDs (Orange bars, n =545) compared with Streptavidin QDots (purple bars, n =188). ( c ) Distribution of diffusion constants of streptavidin-linked SNAP proteins on supported lipid bilayers (thin purple line, n =756, 0.56 μm 2 /s mean) shows a decrease when the concentration of protein is increased 100 fold (thick purple line, n =189, 0.35 μm 2 /s mean). Distribution of diffusion constants of mQD-linked SNAP proteins (thin orange line, n =490, 0.89 μm 2 /s mean) is not altered when protein concentration is increased 100-fold (thick orange line n =790 0.86 μm 2 /s mean). SNAP protein diffusion rates measured with mQDs are nearly identical to diffusion rates measure with small organic dyes such as Atto488 (dotted black line, n =245, 0.89 μm 2 /s mean). Scale bar = 25 nm.

    Techniques Used: Transmission Electron Microscopy, Incubation, Sequencing, Diffusion-based Assay, Concentration Assay, Protein Concentration

    Exclusive synthesis of small, modular, and monovalent quantum dots (QDs) by the principle of Steric Exclusion ( a ) Incubation of bare QDs with trithiol DNA (ttDNA) generates products with a distribution of valencies due to excess nanoparticle surface area. In contrast, phosphorothioate DNA (ptDNA) molecules of appropriate size wrap the nanoparticle, preventing the reaction of a second strand due to Steric Exclusion. ( b ) Agarose gel electrophoresis of reactions of ptDNA and ttDNA of identical length with bare nanoparticles optimized for yield of monovalent products. ( c ) Plot of lambda (average number of molecules bound per QD) versus percent monovalent products using ttDNA and ptDNA. Fitting the curve with a Poisson distribution indicates that the distribution of products generated by ttDNA is underdispersed relative to expected values for large lambda. The same curve for ptDNA is not defined for values of lambda greater than one. (inset) Plot of reaction stoichiometry (ptDNA:QD) versus percent monovalent products. ( d ) Steric Exclusion using 50 adenosine ptDNA sequences efficiently generated monovalent nanoparticles of distinct sizes, shapes, and hence spectral properties. ( e ) Dynamic light scattering analysis reveals that ptDNA-wrapped mQDs are 12 nm in diameter, similar in size to an IgG (dotted line) and about half the size of conventional Streptavidin QDots (22 nm). ( f ) DNA-wrapped mQDs can be selectively targeted by 3’-modification of the oligonucleotide. Alternatively, complementary strands bearing a 5’ targeting modification such as benzylguanine (BG), benzylcytosine (BC) or lipid allow modular targeting of mQDs to streptavidin, SNAP-, CLIP-tags, or cell surfaces.
    Figure Legend Snippet: Exclusive synthesis of small, modular, and monovalent quantum dots (QDs) by the principle of Steric Exclusion ( a ) Incubation of bare QDs with trithiol DNA (ttDNA) generates products with a distribution of valencies due to excess nanoparticle surface area. In contrast, phosphorothioate DNA (ptDNA) molecules of appropriate size wrap the nanoparticle, preventing the reaction of a second strand due to Steric Exclusion. ( b ) Agarose gel electrophoresis of reactions of ptDNA and ttDNA of identical length with bare nanoparticles optimized for yield of monovalent products. ( c ) Plot of lambda (average number of molecules bound per QD) versus percent monovalent products using ttDNA and ptDNA. Fitting the curve with a Poisson distribution indicates that the distribution of products generated by ttDNA is underdispersed relative to expected values for large lambda. The same curve for ptDNA is not defined for values of lambda greater than one. (inset) Plot of reaction stoichiometry (ptDNA:QD) versus percent monovalent products. ( d ) Steric Exclusion using 50 adenosine ptDNA sequences efficiently generated monovalent nanoparticles of distinct sizes, shapes, and hence spectral properties. ( e ) Dynamic light scattering analysis reveals that ptDNA-wrapped mQDs are 12 nm in diameter, similar in size to an IgG (dotted line) and about half the size of conventional Streptavidin QDots (22 nm). ( f ) DNA-wrapped mQDs can be selectively targeted by 3’-modification of the oligonucleotide. Alternatively, complementary strands bearing a 5’ targeting modification such as benzylguanine (BG), benzylcytosine (BC) or lipid allow modular targeting of mQDs to streptavidin, SNAP-, CLIP-tags, or cell surfaces.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Generated, Modification, Cross-linking Immunoprecipitation

    70) Product Images from "The full amino acid repertoire is superior to serine/tyrosine for selection of high affinity immunoglobulin G binders from the fibronectin scaffold"

    Article Title: The full amino acid repertoire is superior to serine/tyrosine for selection of high affinity immunoglobulin G binders from the fibronectin scaffold

    Journal: Protein Engineering, Design and Selection

    doi: 10.1093/protein/gzp083

    Affinity purification. Goat ( a ) or rabbit ( b ) serum was purified on an affinity column composed of streptavidin–agarose and biotinylated Fn3 (gI2.5.3T88I or rI4.5.5K27S/K56S). The serum (S), flowthrough (F), washes (W1–W3) and elution (E) were separated by polyacrylamide gel electrophoresis and stained with SimplyBlue SafeStain. L indicates the protein ladder.
    Figure Legend Snippet: Affinity purification. Goat ( a ) or rabbit ( b ) serum was purified on an affinity column composed of streptavidin–agarose and biotinylated Fn3 (gI2.5.3T88I or rI4.5.5K27S/K56S). The serum (S), flowthrough (F), washes (W1–W3) and elution (E) were separated by polyacrylamide gel electrophoresis and stained with SimplyBlue SafeStain. L indicates the protein ladder.

    Techniques Used: Affinity Purification, Purification, Affinity Column, Polyacrylamide Gel Electrophoresis, Staining

    71) Product Images from "Identification of the Lateral Interaction Surfaces of Human Histocompatibility Leukocyte Antigen (HLA)-DM with HLA-DR1 by Formation of Tethered Complexes That Present Enhanced HLA-DM Catalysis"

    Article Title: Identification of the Lateral Interaction Surfaces of Human Histocompatibility Leukocyte Antigen (HLA)-DM with HLA-DR1 by Formation of Tethered Complexes That Present Enhanced HLA-DM Catalysis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20020117

    Summary of half-lives of intrinsic peptide dissociation for the DM–peptide/DR1 complexes, and Streptavidin/peptide/DR1 constructs. Standard deviations indicated are a result of 3–5 different measurements. On the left is a cartoon representation of the constructs, where the solid thick line represents the peptide bound on DR1 and the thin line the linker between the peptide and DM or Streptavidin. As the dissociation of the complex where the peptide is linked to DM through its COOH terminus is dependent on the concentration ( Fig. 6 ), the half-life shown here is calculated by extrapolating the concentration dependence to infinite dilution (intercept with the y-axis in Fig. 6 ). The half lives or dissociation of the streptavidin/DR1 complexes were estimated from gels F–H of Fig. 4 .
    Figure Legend Snippet: Summary of half-lives of intrinsic peptide dissociation for the DM–peptide/DR1 complexes, and Streptavidin/peptide/DR1 constructs. Standard deviations indicated are a result of 3–5 different measurements. On the left is a cartoon representation of the constructs, where the solid thick line represents the peptide bound on DR1 and the thin line the linker between the peptide and DM or Streptavidin. As the dissociation of the complex where the peptide is linked to DM through its COOH terminus is dependent on the concentration ( Fig. 6 ), the half-life shown here is calculated by extrapolating the concentration dependence to infinite dilution (intercept with the y-axis in Fig. 6 ). The half lives or dissociation of the streptavidin/DR1 complexes were estimated from gels F–H of Fig. 4 .

    Techniques Used: Construct, Concentration Assay

    Purification and analysis of the DM–peptide/DR1 complex. (Top panel) DR1 preloaded with bYHA peptide was mixed with a fourfold excess of streptavidin and then with a 1.5-fold excess of DM–peptide. The mixture was incubated overnight and separated on Sephadex S200 (Amersham Pharmacia Biotech). Positions of molecular weight standards (Bio-Rad Laboratories) are indicated with arrows bellow the chromatogram. (Inset) SDS-PAGE showing the composition of the purified complex compared with equimolar amounts of DM and DR1 controls. Samples were heated to 100°C for 5 min before loading. (Bottom panel) Analysis of DM-pepitde/DR1 complex; SDS-PAGE (4–20%), nonreducing, nonboiled. Lane M, prestained molecular weight standards (Bio-Rad Laboratories). Lane 1, previously purified DM–peptide/DR complex. Lane 2, DR1/bYHA. Lane 3, purified DM. Lane 4, streptavidin. Lane 5, fraction no. 43 (peak 1) from above. Lane 6, fraction no. 48 (peak 2). Lane 7, fraction no. 53 (peak 3). Lane 8, fraction no. 61 (peak 4). Lane 9, fraction no. 73 (peak 5). Note that the DM–peptide/DR1 complex is SDS resistant (lane 1 or lane 5) when the sample is not boiled but falls apart to the DM–peptide and DR1 components when the sample is boiled (inset in chromatogram).
    Figure Legend Snippet: Purification and analysis of the DM–peptide/DR1 complex. (Top panel) DR1 preloaded with bYHA peptide was mixed with a fourfold excess of streptavidin and then with a 1.5-fold excess of DM–peptide. The mixture was incubated overnight and separated on Sephadex S200 (Amersham Pharmacia Biotech). Positions of molecular weight standards (Bio-Rad Laboratories) are indicated with arrows bellow the chromatogram. (Inset) SDS-PAGE showing the composition of the purified complex compared with equimolar amounts of DM and DR1 controls. Samples were heated to 100°C for 5 min before loading. (Bottom panel) Analysis of DM-pepitde/DR1 complex; SDS-PAGE (4–20%), nonreducing, nonboiled. Lane M, prestained molecular weight standards (Bio-Rad Laboratories). Lane 1, previously purified DM–peptide/DR complex. Lane 2, DR1/bYHA. Lane 3, purified DM. Lane 4, streptavidin. Lane 5, fraction no. 43 (peak 1) from above. Lane 6, fraction no. 48 (peak 2). Lane 7, fraction no. 53 (peak 3). Lane 8, fraction no. 61 (peak 4). Lane 9, fraction no. 73 (peak 5). Note that the DM–peptide/DR1 complex is SDS resistant (lane 1 or lane 5) when the sample is not boiled but falls apart to the DM–peptide and DR1 components when the sample is boiled (inset in chromatogram).

    Techniques Used: Purification, Incubation, Molecular Weight, SDS Page

    Time course of the disassembly of DM/DR1 and streptavidin/DR1 complexes followed by native gel electrophoresis. (Left column) Cartoon representations of complex tested. (Right column) Native gel showing time points of the dissociation of each complex upon the addition of excess free HA peptide. In every case the top most band (or bands) corresponds to the DM/DR complex (gels A–E) or the streptavidin/DR complex (gels F–H). Note the difference of dissociation rate between gels A–C and E, and the effect of the solution pH on the dissociation rate in gel D. Complex analyzed is indicated at the bottom of each gel (“/” indicates a noncovalent interaction, where “−” indicates a covalent interaction. The NH 2 terminus of the peptide is considered to be on its left and the COOH terminus on its right).
    Figure Legend Snippet: Time course of the disassembly of DM/DR1 and streptavidin/DR1 complexes followed by native gel electrophoresis. (Left column) Cartoon representations of complex tested. (Right column) Native gel showing time points of the dissociation of each complex upon the addition of excess free HA peptide. In every case the top most band (or bands) corresponds to the DM/DR complex (gels A–E) or the streptavidin/DR complex (gels F–H). Note the difference of dissociation rate between gels A–C and E, and the effect of the solution pH on the dissociation rate in gel D. Complex analyzed is indicated at the bottom of each gel (“/” indicates a noncovalent interaction, where “−” indicates a covalent interaction. The NH 2 terminus of the peptide is considered to be on its left and the COOH terminus on its right).

    Techniques Used: Nucleic Acid Electrophoresis

    Analysis of different methods of assembly of DM–peptide/DR1 complexes by native PAGE. (A) DM–peptide/DR1 complex can be assembled either by loading of DM–peptide onto insect cell produced empty DR1 or by competing out a preloaded CLIP peptide. DM–peptide adduct was mixed either with purified DR1-CLIP or with insect cell produced unloaded DR1, and incubated for 3 d at 25°C. The samples were analyzed on 10% native PAGE. Lane 1, DM; lane 2, DR1/CLIP; lane 3, DM–peptide adduct; lane 4, DM–peptide/DR1-CLIP mixture after incubation; lane 5, DM–peptide/insect DR1 mixture after incubation. (B) Exchange of bYHA peptide bound on DR1 by free HA peptide is accelerated by the binding of streptavidin. Lane 1, streptavidin; lane 2, DR1/bYHA; lane 3, streptavidin mixed with DR1/bYHA; lane 4, DR1/bYHA mixed with HA peptide; lane 5, streptavidin and HA peptide mixed with DR1/bYHA; lanes 6 and 7, DR1/bYHA mixed with HA and incubated at 37°C for 1 h; lane 8, DR1/HA. (C) The DM–peptide/DR1 complex can be assembled with high yield by competing out prebound bYHA peptide on DR1 with the aid of streptavidin. DR1 loaded with the bYHA peptide was mixed with a twofold excess streptavidin and a 1.5-fold excess DM–peptide adduct. The sample was split in two and was either incubated for 10 min or 24 h at 25°C . Lane 1, DM–peptide; lane 2, DR1/bYHA; lane 3, DM–peptide (different preparation); lane 4, DM; lane 5, purified DM–peptide/DR1 complex; lane 6, mixture after 10 min incubation; lane 7, mixture after 24 h incubation.
    Figure Legend Snippet: Analysis of different methods of assembly of DM–peptide/DR1 complexes by native PAGE. (A) DM–peptide/DR1 complex can be assembled either by loading of DM–peptide onto insect cell produced empty DR1 or by competing out a preloaded CLIP peptide. DM–peptide adduct was mixed either with purified DR1-CLIP or with insect cell produced unloaded DR1, and incubated for 3 d at 25°C. The samples were analyzed on 10% native PAGE. Lane 1, DM; lane 2, DR1/CLIP; lane 3, DM–peptide adduct; lane 4, DM–peptide/DR1-CLIP mixture after incubation; lane 5, DM–peptide/insect DR1 mixture after incubation. (B) Exchange of bYHA peptide bound on DR1 by free HA peptide is accelerated by the binding of streptavidin. Lane 1, streptavidin; lane 2, DR1/bYHA; lane 3, streptavidin mixed with DR1/bYHA; lane 4, DR1/bYHA mixed with HA peptide; lane 5, streptavidin and HA peptide mixed with DR1/bYHA; lanes 6 and 7, DR1/bYHA mixed with HA and incubated at 37°C for 1 h; lane 8, DR1/HA. (C) The DM–peptide/DR1 complex can be assembled with high yield by competing out prebound bYHA peptide on DR1 with the aid of streptavidin. DR1 loaded with the bYHA peptide was mixed with a twofold excess streptavidin and a 1.5-fold excess DM–peptide adduct. The sample was split in two and was either incubated for 10 min or 24 h at 25°C . Lane 1, DM–peptide; lane 2, DR1/bYHA; lane 3, DM–peptide (different preparation); lane 4, DM; lane 5, purified DM–peptide/DR1 complex; lane 6, mixture after 10 min incubation; lane 7, mixture after 24 h incubation.

    Techniques Used: Clear Native PAGE, Produced, Cross-linking Immunoprecipitation, Purification, Incubation, Binding Assay

    72) Product Images from "Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy"

    Article Title: Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy

    Journal: eLife

    doi: 10.7554/eLife.05413

    Detection of the transient state with a molecular sized fluorescent label. ( A ) Representative distance time series of a myosin 5 molecule labelled with an atto-647N/streptavidin conjugate at the N-terminus and tracked using single molecule total internal fluorescence microscopy. ( B ) Corresponding 2D trajectory. ( C ) Additional traces exhibiting the transient state marked by arrows. ATP concentration: 10 μM. Scale bar: 50 nm. Imaging speed: 100 frames/s. DOI: http://dx.doi.org/10.7554/eLife.05413.005
    Figure Legend Snippet: Detection of the transient state with a molecular sized fluorescent label. ( A ) Representative distance time series of a myosin 5 molecule labelled with an atto-647N/streptavidin conjugate at the N-terminus and tracked using single molecule total internal fluorescence microscopy. ( B ) Corresponding 2D trajectory. ( C ) Additional traces exhibiting the transient state marked by arrows. ATP concentration: 10 μM. Scale bar: 50 nm. Imaging speed: 100 frames/s. DOI: http://dx.doi.org/10.7554/eLife.05413.005

    Techniques Used: Fluorescence, Microscopy, Concentration Assay, Imaging

    Additional traces of simultaneous scattering and fluorescence tracking of a single myosin 5a. One head was labelled with a 20 nm streptavidin-functionalized gold particle (red) and the other with a fluorescent quantum dot (blue). Reported values correspond to the standard deviation σ in the position of the bound state (nm) and the shaded regions encompass an area of 3σ. The traces are colored according to the label colors in the inset. ATP concentration: 10 μM. Scale bar: 100 nm. Imaging speed: 100 frames/s. DOI: http://dx.doi.org/10.7554/eLife.05413.011
    Figure Legend Snippet: Additional traces of simultaneous scattering and fluorescence tracking of a single myosin 5a. One head was labelled with a 20 nm streptavidin-functionalized gold particle (red) and the other with a fluorescent quantum dot (blue). Reported values correspond to the standard deviation σ in the position of the bound state (nm) and the shaded regions encompass an area of 3σ. The traces are colored according to the label colors in the inset. ATP concentration: 10 μM. Scale bar: 100 nm. Imaging speed: 100 frames/s. DOI: http://dx.doi.org/10.7554/eLife.05413.011

    Techniques Used: Fluorescence, Standard Deviation, Concentration Assay, Imaging

    73) Product Images from "Resolving molecule-specific information in dynamic lipid membrane processes with multi-resonant infrared metasurfaces"

    Article Title: Resolving molecule-specific information in dynamic lipid membrane processes with multi-resonant infrared metasurfaces

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04594-x

    Simultaneous monitoring of multiple biological analytes. a Schematic of the experimental configuration. b Infrared reflectance spectrum of the multi-resonant sensor chip in PBS buffer solution. c Reflectance spectra before ( R 0 ) and after ( R ) lipid membrane formation in the CH 2 band spectral region, magnified from marked area in ( b ). d Differential absorption spectrum calculated from the reflectance spectra in ( c ). The dashed line corresponds to the second-order polynomial used for baseline correction. e Color-coded time-dependent differential absorption spectra acquired during the lipid membrane formation and streptavidin-binding experiment. f Time trace of the integrated absorbance signal in the amide (red-shaded area) and CH 2 (green-shaded area) bands from ( e ). The lipid and streptadivin injection steps are indicated by the blue- and orange-shaded areas, respectively. The integrated absorbance signals from the amide (red curve) and CH 2 (green curve) bands exhibit pronounced signal modulations during the lipid membrane formation and streptavidin-binding steps, evidencing an inadequate discrimination of the two analytes. g Reference spectra for the lipid (blue-shaded area) and streptavidin (orange-shaded area) signal contributions. h Linear regression signals obtained from the spectral data in ( e ) with respect to the reference spectra in ( g ). Linear regression signals for lipid (blue curve) and streptadivin (orange curve) show a significant signal increase only during the corresponding lipid or streptavidin injection step, demonstrating effective chemical discrimination
    Figure Legend Snippet: Simultaneous monitoring of multiple biological analytes. a Schematic of the experimental configuration. b Infrared reflectance spectrum of the multi-resonant sensor chip in PBS buffer solution. c Reflectance spectra before ( R 0 ) and after ( R ) lipid membrane formation in the CH 2 band spectral region, magnified from marked area in ( b ). d Differential absorption spectrum calculated from the reflectance spectra in ( c ). The dashed line corresponds to the second-order polynomial used for baseline correction. e Color-coded time-dependent differential absorption spectra acquired during the lipid membrane formation and streptavidin-binding experiment. f Time trace of the integrated absorbance signal in the amide (red-shaded area) and CH 2 (green-shaded area) bands from ( e ). The lipid and streptadivin injection steps are indicated by the blue- and orange-shaded areas, respectively. The integrated absorbance signals from the amide (red curve) and CH 2 (green curve) bands exhibit pronounced signal modulations during the lipid membrane formation and streptavidin-binding steps, evidencing an inadequate discrimination of the two analytes. g Reference spectra for the lipid (blue-shaded area) and streptavidin (orange-shaded area) signal contributions. h Linear regression signals obtained from the spectral data in ( e ) with respect to the reference spectra in ( g ). Linear regression signals for lipid (blue curve) and streptadivin (orange curve) show a significant signal increase only during the corresponding lipid or streptavidin injection step, demonstrating effective chemical discrimination

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Injection

    74) Product Images from "Inhibition of human retinal pigment epithelial cell attachment, spreading, and migration by the human lectin galectin-1"

    Article Title: Inhibition of human retinal pigment epithelial cell attachment, spreading, and migration by the human lectin galectin-1

    Journal: Molecular Vision

    doi:

    Effects of galectin-1 on the morphology of cultured human retinal pigment epithelial cells and cell surface binding of galectin-1. A-D : Human retinal pigment epithelial (RPE) cell suspensions were preincubated for 35 min without galectin-1 ( A ), or with 125 μg/ml galectin-1 ( B ), or 100 mM β-lactose before addition of 125 µg/ml galectin-1 ( C ), or 100 mM β-lactose ( D ) in the medium. RPE cells were then plated at a density of 0.5×10 4 cells per well in 96-well plates and allowed to adhere for 90 min ( A - D ). The cells were observed by light microscopy (magnification 100×). E-H : Galectin-1 is detected on the surface of human RPE cells by immunofluorescence. Cells were cultured on glass coverslips for 16 h before being treated with biotinylated galectin-1 ( E ). They were fixed, then stained with a fluorescent streptavidin conjugate. For controls ( G ), untreated cells were exposed to streptavidin conjugate alone. Nuclei were counterstained with 1 µg/ml Hoechst 33342 ( F , H ). Localization of bound galectin-1 was visualized by fluorescence microscopy at a 400x magnification. The bar represents 100 μm.
    Figure Legend Snippet: Effects of galectin-1 on the morphology of cultured human retinal pigment epithelial cells and cell surface binding of galectin-1. A-D : Human retinal pigment epithelial (RPE) cell suspensions were preincubated for 35 min without galectin-1 ( A ), or with 125 μg/ml galectin-1 ( B ), or 100 mM β-lactose before addition of 125 µg/ml galectin-1 ( C ), or 100 mM β-lactose ( D ) in the medium. RPE cells were then plated at a density of 0.5×10 4 cells per well in 96-well plates and allowed to adhere for 90 min ( A - D ). The cells were observed by light microscopy (magnification 100×). E-H : Galectin-1 is detected on the surface of human RPE cells by immunofluorescence. Cells were cultured on glass coverslips for 16 h before being treated with biotinylated galectin-1 ( E ). They were fixed, then stained with a fluorescent streptavidin conjugate. For controls ( G ), untreated cells were exposed to streptavidin conjugate alone. Nuclei were counterstained with 1 µg/ml Hoechst 33342 ( F , H ). Localization of bound galectin-1 was visualized by fluorescence microscopy at a 400x magnification. The bar represents 100 μm.

    Techniques Used: Cell Culture, Binding Assay, Light Microscopy, Immunofluorescence, Staining, Fluorescence, Microscopy

    75) Product Images from "Working with Commercially Available Quantum Dots for Immunofluorescence on Tissue Sections"

    Article Title: Working with Commercially Available Quantum Dots for Immunofluorescence on Tissue Sections

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163856

    Decreased immunofluorescence during multiplex staining. Serial sections were stained with 1 to 5 different markers (Marker 1, MHCII Qdot 705Vivid; Marker 2, HO1 Qdot 605Vivid; Marker 3, CD163 Qdot 565; Marker 4, CD206 Qdot 655Vivid; Marker 5, CD68 streptavidin Qdot 525). After each staining round, a slide was mounted and imaged. Analysis of the intensity of Qdots 705Vivid, 605Vivid and 565 fluorescence is shown (Qdot 655Vivid 525 in S3 Fig ) for one representative field (aligned in serial sections). Each dot corresponds to one thresholded positive cell. The intensity of fluorescence is affected by the number of staining rounds (p
    Figure Legend Snippet: Decreased immunofluorescence during multiplex staining. Serial sections were stained with 1 to 5 different markers (Marker 1, MHCII Qdot 705Vivid; Marker 2, HO1 Qdot 605Vivid; Marker 3, CD163 Qdot 565; Marker 4, CD206 Qdot 655Vivid; Marker 5, CD68 streptavidin Qdot 525). After each staining round, a slide was mounted and imaged. Analysis of the intensity of Qdots 705Vivid, 605Vivid and 565 fluorescence is shown (Qdot 655Vivid 525 in S3 Fig ) for one representative field (aligned in serial sections). Each dot corresponds to one thresholded positive cell. The intensity of fluorescence is affected by the number of staining rounds (p

    Techniques Used: Immunofluorescence, Multiplex Assay, Staining, Marker, Fluorescence

    Quenching of Vivid but not original Qdots under LED fluorescence illumination. FFPE serial liver sections were labelled for the marker CD68 using a Labelled StrepAvidin Biotin (LSAB) method with either original streptavidin conjugated Qdot605 or streptavidin conjugated Vivid Qdot605. Fig 2A shows the intensity of fluorescence for one representative cell stained with either the original Qdot (left) or the Vivid Qdot (right): t0 represents the initial capture of the image; the sections were then left under constant illumination with LED 425nm for 2 minutes and finally left for a further 2 minutes in the dark. Note the difference in the intensity scale 0–5.95 for Original Qdot and 0–10.40 for Vivid Qdot. Fig 2B: Intensity profile along the line drawn on each image. Line profiles were drawn on the initial images (t0) using the Nuance software and copied onto the other images. For both original (left) and Vivid Qdot605 (right), the line profile intensities are given scaled to max (red lines: initial intensity; green lines: after 2 minutes illumination with LED 325nm, blue after a further 2 minutes in the dark). The scale is given by the lines which all are 13 μm in length.
    Figure Legend Snippet: Quenching of Vivid but not original Qdots under LED fluorescence illumination. FFPE serial liver sections were labelled for the marker CD68 using a Labelled StrepAvidin Biotin (LSAB) method with either original streptavidin conjugated Qdot605 or streptavidin conjugated Vivid Qdot605. Fig 2A shows the intensity of fluorescence for one representative cell stained with either the original Qdot (left) or the Vivid Qdot (right): t0 represents the initial capture of the image; the sections were then left under constant illumination with LED 425nm for 2 minutes and finally left for a further 2 minutes in the dark. Note the difference in the intensity scale 0–5.95 for Original Qdot and 0–10.40 for Vivid Qdot. Fig 2B: Intensity profile along the line drawn on each image. Line profiles were drawn on the initial images (t0) using the Nuance software and copied onto the other images. For both original (left) and Vivid Qdot605 (right), the line profile intensities are given scaled to max (red lines: initial intensity; green lines: after 2 minutes illumination with LED 325nm, blue after a further 2 minutes in the dark). The scale is given by the lines which all are 13 μm in length.

    Techniques Used: Fluorescence, Formalin-fixed Paraffin-Embedded, Marker, Staining, Software

    Related Articles

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    Synthesized:

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    TA Cloning:

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    Electrophoresis:

    Article Title: N-glycosylation of ICAM-2 is required for ICAM-2-mediated complete suppression of metastatic potential of SK-N-AS neuroblastoma cells
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    Incubation:

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    Stripping Membranes:

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    Western Blot:

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    Transfection:

    Article Title: N-glycosylation of ICAM-2 is required for ICAM-2-mediated complete suppression of metastatic potential of SK-N-AS neuroblastoma cells
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    Article Title: Na+/H+ Exchanger Regulatory Factor 3 Is Critical for Multidrug Resistance Protein 4–Mediated Drug Efflux in the Kidney
    Article Snippet: Briefly, transfected cells were washed with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 , and the plasma membrane proteins were biotinylated by sulfo-NHS-SS-biotin (Pierce) in PBS for 30 minutes at 4°C. .. The cells were lysed, and an avidin-containing solution (Streptavidin Agarose Resins; Pierce) was added to the supernatant; the mixture was incubated at 4°C overnight.

    Chromatography:

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    Concentration Assay:

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    Protease Inhibitor:

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    MTT Assay:

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    Avidin-Biotin Assay:

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    Purification:

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    Staining:

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    SDS Page:

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    Agarose Gel Electrophoresis:

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    Immunoprecipitation:

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    Lysis:

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    Thermo Fisher streptavidin
    The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through <t>biotin–streptavidin</t> interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.
    Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin biotin interactions
    Effect of sequence length on assay. The HRP signals (bars) are expressed as the percentages of the fully cognate positive control (the dark grey bar 40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the <t>streptavidin-modified</t> surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length and end sequence. The Bgl II restriction site is indicated with thick horizontal lines. Target-probe duplex designations indicate the complementary sequence length, or fraction of complementary sequence to the total target length. The target oligonucleotide sequences are shown in Table 1 .
    Streptavidin Biotin Interactions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher streptavidin coated 96 well maxisorp plates
    ), are shown. (B) Peptide A specificity of the MAbs in an ELISA. Biotin-conjugated peptide A was added to <t>streptavidin-coated</t> 96-well plates (200 ng/well). Each MAb (ascites fluid) was diluted 1:1,000 and used as the primary antibody. The y . An ELISA was performed with 1:20,000-diluted ascites fluid in the presence or absence of various concentrations of NaSCN as indicated. The specific binding affinity was calculated based on the values obtained with or without NaSCN. The data shown represent three independent experiments. Error bars represent the standard deviation.
    Streptavidin Coated 96 Well Maxisorp Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin coated 96 well maxisorp plates/product/Thermo Fisher
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    Image Search Results


    The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through biotin–streptavidin interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.

    Journal: Nucleic Acids Research

    Article Title: A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

    doi: 10.1093/nar/gkv069

    Figure Lengend Snippet: The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through biotin–streptavidin interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.

    Article Snippet: Materials Biotin-labeled hairpin DNA oligomer FL814 containing a specific binding site of HMGA2 was purchased from Eurofins MWG Operon, Inc. Streptavidin covalently coated 96-well plates (NUNC Immobilizer Streptavidin-F96 clear) were from Thermo Fisher Scientific, Inc. Antibody against HMGA2 (HMGA2 (D1A7) Rabbit mAb) and Anti-rabbit IgG, HRP-linked Antibody #7074 were purchased from Cell Signaling, Inc. Ultra TMB-ELISA was bought from Thermo Fisher Scientific, Inc.

    Techniques: High Throughput Screening Assay, Labeling, Blocking Assay, Binding Assay, Protein Binding, Fluorescence

    Effect of sequence length on assay. The HRP signals (bars) are expressed as the percentages of the fully cognate positive control (the dark grey bar 40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length and end sequence. The Bgl II restriction site is indicated with thick horizontal lines. Target-probe duplex designations indicate the complementary sequence length, or fraction of complementary sequence to the total target length. The target oligonucleotide sequences are shown in Table 1 .

    Journal: PLoS ONE

    Article Title: A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

    doi: 10.1371/journal.pone.0097826

    Figure Lengend Snippet: Effect of sequence length on assay. The HRP signals (bars) are expressed as the percentages of the fully cognate positive control (the dark grey bar 40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length and end sequence. The Bgl II restriction site is indicated with thick horizontal lines. Target-probe duplex designations indicate the complementary sequence length, or fraction of complementary sequence to the total target length. The target oligonucleotide sequences are shown in Table 1 .

    Article Snippet: Restriction enzyme assay protocol For surface immobilization of the HRP-probe through streptavidin-biotin interactions, 30 µL of 50 nM dilution of MCA-BG-HRP conjugate in PBS (1∶100 dilution of 5 µM stock) was applied to each well of a streptavidin-pre-coated 96-microwell plate (Thermo Scientific).

    Techniques: Sequencing, Positive Control, Modification

    Effects of restriction site positioning within the ds DNA hybrid, and non-complementary loop addition. The HRP signals (bars) are expressed as percentages of the fully cognate positive control (40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length, non-complementary ends, and/or loops. The Bgl II restriction site is indicated with thick horizontal lines. Target designations are the following: 5′ (or 3′), corresponds to the 5′ (or 3′) ends of the full length positive control; C, control (fully cognate), L, loop (addition of 5 or 10 nucleotides); rs5′ (or rs3′), the end of restriction site to which 0, 3, or 5 (+0, +3, +5) complementary nucleotides were added. For rs3′+0, two targets were prepared that had different non-complementary sequences flanking the 3′-end of the restriction site (rs3′+0-A, rs3′+0-G). The target oligonucleotide sequences are shown in Table 1 .

    Journal: PLoS ONE

    Article Title: A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

    doi: 10.1371/journal.pone.0097826

    Figure Lengend Snippet: Effects of restriction site positioning within the ds DNA hybrid, and non-complementary loop addition. The HRP signals (bars) are expressed as percentages of the fully cognate positive control (40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a target of variable length, non-complementary ends, and/or loops. The Bgl II restriction site is indicated with thick horizontal lines. Target designations are the following: 5′ (or 3′), corresponds to the 5′ (or 3′) ends of the full length positive control; C, control (fully cognate), L, loop (addition of 5 or 10 nucleotides); rs5′ (or rs3′), the end of restriction site to which 0, 3, or 5 (+0, +3, +5) complementary nucleotides were added. For rs3′+0, two targets were prepared that had different non-complementary sequences flanking the 3′-end of the restriction site (rs3′+0-A, rs3′+0-G). The target oligonucleotide sequences are shown in Table 1 .

    Article Snippet: Restriction enzyme assay protocol For surface immobilization of the HRP-probe through streptavidin-biotin interactions, 30 µL of 50 nM dilution of MCA-BG-HRP conjugate in PBS (1∶100 dilution of 5 µM stock) was applied to each well of a streptavidin-pre-coated 96-microwell plate (Thermo Scientific).

    Techniques: Positive Control, Modification

    General schematic of the restriction enzyme assay. ( A ) Surface immobilization of HRP conjugated to an oligonucleotide probe specific for a target gene of interest. ( B ) The target DNA (an oligonucleotide or a denatured PCR amplicon) is hybridized to the immobilized probe. ( C ) Addition of a restriction enzyme (Rrec) that recognizes and cleaves the target-probe ds DNA hybrid, resulting in release of the HRP marker into the reaction solution. ( D ) The reaction solution is transferred into a new well and mixed with an HRP substrate for colorimetric detection. For each target DNA molecule one HRP molecule is released, resulting in a linear dependence of the signal on the target DNA concentration. ( E ) Detailed schematic of the double stranded target-probe DNA duplex, with the specific restriction site shown in purple. HRP, horseradish peroxidase; B, biotin; SA, streptavidin.

    Journal: PLoS ONE

    Article Title: A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

    doi: 10.1371/journal.pone.0097826

    Figure Lengend Snippet: General schematic of the restriction enzyme assay. ( A ) Surface immobilization of HRP conjugated to an oligonucleotide probe specific for a target gene of interest. ( B ) The target DNA (an oligonucleotide or a denatured PCR amplicon) is hybridized to the immobilized probe. ( C ) Addition of a restriction enzyme (Rrec) that recognizes and cleaves the target-probe ds DNA hybrid, resulting in release of the HRP marker into the reaction solution. ( D ) The reaction solution is transferred into a new well and mixed with an HRP substrate for colorimetric detection. For each target DNA molecule one HRP molecule is released, resulting in a linear dependence of the signal on the target DNA concentration. ( E ) Detailed schematic of the double stranded target-probe DNA duplex, with the specific restriction site shown in purple. HRP, horseradish peroxidase; B, biotin; SA, streptavidin.

    Article Snippet: Restriction enzyme assay protocol For surface immobilization of the HRP-probe through streptavidin-biotin interactions, 30 µL of 50 nM dilution of MCA-BG-HRP conjugate in PBS (1∶100 dilution of 5 µM stock) was applied to each well of a streptavidin-pre-coated 96-microwell plate (Thermo Scientific).

    Techniques: Enzymatic Assay, Polymerase Chain Reaction, Amplification, Marker, Concentration Assay

    Effect of point mutations introduced into the target sequence. ( A ) Single, double and triple mutations were introduced between the target center and the 3′ end corresponding to the surface-immobilized terminus of the target-probe duplex. ( B ) Mutations were introduced between the target center and the 5′ end corresponding to the end of the target-probe duplex that was free in solution. HRP signals (bars) are expressed as the percentages of the fully cognate positive control (dark grey bar 40, for 40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a 40-mer target with 1–3 mutations shown with black ovals. The Bgl II restriction site is indicated with thick horizontal lines. Targets are named with ‘rs’ for mutations introduced within the restriction site, otherwise the target name contains the replacement nucleotide (mostly G) and position within the sequence, starting from the 5′ target end. The rs19+24 contained two mutations at the ends of the restriction site. Target oligonucleotide sequences are shown in Table 1 .

    Journal: PLoS ONE

    Article Title: A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

    doi: 10.1371/journal.pone.0097826

    Figure Lengend Snippet: Effect of point mutations introduced into the target sequence. ( A ) Single, double and triple mutations were introduced between the target center and the 3′ end corresponding to the surface-immobilized terminus of the target-probe duplex. ( B ) Mutations were introduced between the target center and the 5′ end corresponding to the end of the target-probe duplex that was free in solution. HRP signals (bars) are expressed as the percentages of the fully cognate positive control (dark grey bar 40, for 40-mer). Target-probe duplexes shown below the bars consist of (1) the probe attached to the streptavidin-modified surface with biotin (bottom) and conjugated to HRP (top), and (2) a 40-mer target with 1–3 mutations shown with black ovals. The Bgl II restriction site is indicated with thick horizontal lines. Targets are named with ‘rs’ for mutations introduced within the restriction site, otherwise the target name contains the replacement nucleotide (mostly G) and position within the sequence, starting from the 5′ target end. The rs19+24 contained two mutations at the ends of the restriction site. Target oligonucleotide sequences are shown in Table 1 .

    Article Snippet: Restriction enzyme assay protocol For surface immobilization of the HRP-probe through streptavidin-biotin interactions, 30 µL of 50 nM dilution of MCA-BG-HRP conjugate in PBS (1∶100 dilution of 5 µM stock) was applied to each well of a streptavidin-pre-coated 96-microwell plate (Thermo Scientific).

    Techniques: Sequencing, Positive Control, Modification

    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    ), are shown. (B) Peptide A specificity of the MAbs in an ELISA. Biotin-conjugated peptide A was added to streptavidin-coated 96-well plates (200 ng/well). Each MAb (ascites fluid) was diluted 1:1,000 and used as the primary antibody. The y . An ELISA was performed with 1:20,000-diluted ascites fluid in the presence or absence of various concentrations of NaSCN as indicated. The specific binding affinity was calculated based on the values obtained with or without NaSCN. The data shown represent three independent experiments. Error bars represent the standard deviation.

    Journal: Journal of Virology

    Article Title: Amino Acid Residue-Specific Neutralization and Nonneutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein

    doi: 10.1128/JVI.00994-12

    Figure Lengend Snippet: ), are shown. (B) Peptide A specificity of the MAbs in an ELISA. Biotin-conjugated peptide A was added to streptavidin-coated 96-well plates (200 ng/well). Each MAb (ascites fluid) was diluted 1:1,000 and used as the primary antibody. The y . An ELISA was performed with 1:20,000-diluted ascites fluid in the presence or absence of various concentrations of NaSCN as indicated. The specific binding affinity was calculated based on the values obtained with or without NaSCN. The data shown represent three independent experiments. Error bars represent the standard deviation.

    Article Snippet: Biotin-conjugated peptide (200 ng/well) was added to streptavidin-coated 96-well Maxisorp plates (Thermo Fisher Scientific, Rockford, IL), followed by incubation at room temperature for 1 h in Super Block blocking buffer (Thermo Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Standard Deviation

    Use of mutational analysis to identify the residues that are critical for antibody recognition. (A) Biotin-conjugated peptides were chemically synthesized to represent the truncated peptide B, i.e., B short, from the E2 protein of HCV genotype 1a (H77 strain) and its mutations. The B short mutant peptides contained a single alanine substitution at positions 437, 438, 440, 441, and 442, respectively. A hyphen indicates an amino acid residue identical to that of the H77 sequence. (B) Biotin-conjugated B short peptide and its mutants were added to streptavidin-coated 96-well plates at 200 ng/well in an ELISA. Each MAb (ascites fluid) was diluted 1:10 5 dilution, and applied as the primary antibody. The data shown represent at least three independent experiments. The x axis indicates the antibodies used in the assay. The y axis indicates the absorbance at 405 nm, representing specific binding of a given antibody to each individual peptide.

    Journal: Journal of Virology

    Article Title: Amino Acid Residue-Specific Neutralization and Nonneutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein

    doi: 10.1128/JVI.00994-12

    Figure Lengend Snippet: Use of mutational analysis to identify the residues that are critical for antibody recognition. (A) Biotin-conjugated peptides were chemically synthesized to represent the truncated peptide B, i.e., B short, from the E2 protein of HCV genotype 1a (H77 strain) and its mutations. The B short mutant peptides contained a single alanine substitution at positions 437, 438, 440, 441, and 442, respectively. A hyphen indicates an amino acid residue identical to that of the H77 sequence. (B) Biotin-conjugated B short peptide and its mutants were added to streptavidin-coated 96-well plates at 200 ng/well in an ELISA. Each MAb (ascites fluid) was diluted 1:10 5 dilution, and applied as the primary antibody. The data shown represent at least three independent experiments. The x axis indicates the antibodies used in the assay. The y axis indicates the absorbance at 405 nm, representing specific binding of a given antibody to each individual peptide.

    Article Snippet: Biotin-conjugated peptide (200 ng/well) was added to streptavidin-coated 96-well Maxisorp plates (Thermo Fisher Scientific, Rockford, IL), followed by incubation at room temperature for 1 h in Super Block blocking buffer (Thermo Scientific).

    Techniques: Synthesized, Mutagenesis, Sequencing, Enzyme-linked Immunosorbent Assay, Binding Assay