streptavidin  (Millipore)


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    Structured Review

    Millipore streptavidin
    Schematic of the sandwich-assays using two types of functionalized NPs for the detection of CEA in the three different conditions studied in this paper. (A) Assay: Primary antibody immobilized on the sensor surface, incubated with EA in PBS BSA and either NP functionalized with the secondary antibody specific for CEA (Ab 2 -NPs) or secondary antibody and NPs functionalized with <t>streptavidin</t> (S-NPs) binding to the biotinylated Ab 2 B. (B) Assay: Analogous to (A) except for that the sensor is exposed to plasma after CEA capture. (C) Assay: Analogous to (A) except for that the functionalized NPs are contained in blood plasma.
    Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Peptide Functionalization of Gold Nanoparticles for the Detection of Carcinoembryonic Antigen in Blood Plasma via SPR-Based Biosensor"

    Article Title: Peptide Functionalization of Gold Nanoparticles for the Detection of Carcinoembryonic Antigen in Blood Plasma via SPR-Based Biosensor

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2019.00040

    Schematic of the sandwich-assays using two types of functionalized NPs for the detection of CEA in the three different conditions studied in this paper. (A) Assay: Primary antibody immobilized on the sensor surface, incubated with EA in PBS BSA and either NP functionalized with the secondary antibody specific for CEA (Ab 2 -NPs) or secondary antibody and NPs functionalized with streptavidin (S-NPs) binding to the biotinylated Ab 2 B. (B) Assay: Analogous to (A) except for that the sensor is exposed to plasma after CEA capture. (C) Assay: Analogous to (A) except for that the functionalized NPs are contained in blood plasma.
    Figure Legend Snippet: Schematic of the sandwich-assays using two types of functionalized NPs for the detection of CEA in the three different conditions studied in this paper. (A) Assay: Primary antibody immobilized on the sensor surface, incubated with EA in PBS BSA and either NP functionalized with the secondary antibody specific for CEA (Ab 2 -NPs) or secondary antibody and NPs functionalized with streptavidin (S-NPs) binding to the biotinylated Ab 2 B. (B) Assay: Analogous to (A) except for that the sensor is exposed to plasma after CEA capture. (C) Assay: Analogous to (A) except for that the functionalized NPs are contained in blood plasma.

    Techniques Used: Incubation, Binding Assay, Capture-C

    2) Product Images from "Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2"

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt315

    ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of streptavidin (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).
    Figure Legend Snippet: ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of streptavidin (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).

    Techniques Used: Binding Assay, Agarose Gel Electrophoresis, Incubation, Blocking Assay, Staining

    3) Product Images from "Upstream Stimulatory Factors 1 and 2 Mediate the Transcription of Angiotensin II Binding and Inhibitory Protein *"

    Article Title: Upstream Stimulatory Factors 1 and 2 Mediate the Transcription of Angiotensin II Binding and Inhibitory Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.451054

    Identification of Usf1 and Usf2 interaction with the E-box (−72 to −43) of the mouse Agtrap promoter by streptavidin-biotin complex assay and ChIP assay. A , streptavidin-biotin complex assay. Nuclear extracts from mDCT cells were incubated with streptavidin immobilized on agarose beads. The streptavidin-biotin-DNA complex was eluted with SDS buffer and visualized by immunoblot analysis. E-b, E-box biotin-labeled probe; E-n, E-box nonlabeled probe; X-b, X-box biotin-labeled probe; X-n, X-box nonlabeled probe; NP, nuclear extracts; Cy, cytosolic extracts. B , ChIP assay. The mDCT cells were treated with an anti-USF1 antibody, anti-USF2 antibody, anti-SREBP1 antibody, anti-BMAL1 antibody, or control IgG (rabbit anti-HA antibody). Co-immunoprecipitated DNA was purified and estimated by quantitative PCR. In the upper panel the relative amount of DNA fragment detected per antibody is shown. In the lower panel , the quantitative PCR products, which were loaded on 3% agarose gels and visualized by ethidium bromide staining, are shown. Experiments were independently repeated at least three times, and data are expressed as the means ± S.E. *, p
    Figure Legend Snippet: Identification of Usf1 and Usf2 interaction with the E-box (−72 to −43) of the mouse Agtrap promoter by streptavidin-biotin complex assay and ChIP assay. A , streptavidin-biotin complex assay. Nuclear extracts from mDCT cells were incubated with streptavidin immobilized on agarose beads. The streptavidin-biotin-DNA complex was eluted with SDS buffer and visualized by immunoblot analysis. E-b, E-box biotin-labeled probe; E-n, E-box nonlabeled probe; X-b, X-box biotin-labeled probe; X-n, X-box nonlabeled probe; NP, nuclear extracts; Cy, cytosolic extracts. B , ChIP assay. The mDCT cells were treated with an anti-USF1 antibody, anti-USF2 antibody, anti-SREBP1 antibody, anti-BMAL1 antibody, or control IgG (rabbit anti-HA antibody). Co-immunoprecipitated DNA was purified and estimated by quantitative PCR. In the upper panel the relative amount of DNA fragment detected per antibody is shown. In the lower panel , the quantitative PCR products, which were loaded on 3% agarose gels and visualized by ethidium bromide staining, are shown. Experiments were independently repeated at least three times, and data are expressed as the means ± S.E. *, p

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Labeling, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Staining

    4) Product Images from "Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation"

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2009.06935.x

    Assaying the cleavage of single-stranded segments linked via conjugation. A. The binding of 5′-biotinylated LU13 to streptavidin as monitored using native gel electrophoresis. The position of streptavidin was detected by staining with Coomassie blue. Labelling on the right indicates the position of unbound streptavidin (U) and streptavidin bound to 5′-biotinylated LU13 (B). Lanes 1–4 correspond to oligonucleotide amounts of 0.3, 0.6, 1.2 and 1.5 nmol respectively. The amount of streptavidin was 0.15 nmol in all the conjugation reactions. Lane S contains only streptavidin. 5′-hydroxylated LU13 was included as a control. B. The results of incubating 5′-biotinylated (Biotin) and hydroxylated (HO) LU13 with NTH-RNase E after mixing a fourfold molar excess with streptavidin (A). Lanes 1–5 correspond to samples removed after incubating with enzyme for 0, 5, 15, 30 and 60 min. The enzyme and substrate concentrations were 5 and 65 nM respectively. Lanes 6 and 7 correspond to samples incubated in reaction buffer without enzyme for 0 and 60 min respectively. The samples were separated on a denaturing 15% (w/v) polyacrylamide gel. C. Plots of the amount of product formed with time for the reactions shown in (B) and controls that were not mixed with streptavidin prior to incubating with NTH-RNase E (original gels not shown). Open and closed circles correspond to 5′-biotinylated and hydroxylated LU13, respectively, which had been preincubated with streptavidin, while the open and closed squares correspond to incubation of these substrates directly with NTH-RNase E. Closed triangles correspond to prewarmed 5′-monophosphorylated BR13. The substrate concentration was reduced to 65 nM (cf. Fig. 1 ) to slow the reaction, thereby permitting easier comparison with the 5′-monophosphorylated BR13 control.
    Figure Legend Snippet: Assaying the cleavage of single-stranded segments linked via conjugation. A. The binding of 5′-biotinylated LU13 to streptavidin as monitored using native gel electrophoresis. The position of streptavidin was detected by staining with Coomassie blue. Labelling on the right indicates the position of unbound streptavidin (U) and streptavidin bound to 5′-biotinylated LU13 (B). Lanes 1–4 correspond to oligonucleotide amounts of 0.3, 0.6, 1.2 and 1.5 nmol respectively. The amount of streptavidin was 0.15 nmol in all the conjugation reactions. Lane S contains only streptavidin. 5′-hydroxylated LU13 was included as a control. B. The results of incubating 5′-biotinylated (Biotin) and hydroxylated (HO) LU13 with NTH-RNase E after mixing a fourfold molar excess with streptavidin (A). Lanes 1–5 correspond to samples removed after incubating with enzyme for 0, 5, 15, 30 and 60 min. The enzyme and substrate concentrations were 5 and 65 nM respectively. Lanes 6 and 7 correspond to samples incubated in reaction buffer without enzyme for 0 and 60 min respectively. The samples were separated on a denaturing 15% (w/v) polyacrylamide gel. C. Plots of the amount of product formed with time for the reactions shown in (B) and controls that were not mixed with streptavidin prior to incubating with NTH-RNase E (original gels not shown). Open and closed circles correspond to 5′-biotinylated and hydroxylated LU13, respectively, which had been preincubated with streptavidin, while the open and closed squares correspond to incubation of these substrates directly with NTH-RNase E. Closed triangles correspond to prewarmed 5′-monophosphorylated BR13. The substrate concentration was reduced to 65 nM (cf. Fig. 1 ) to slow the reaction, thereby permitting easier comparison with the 5′-monophosphorylated BR13 control.

    Techniques Used: Conjugation Assay, Binding Assay, Nucleic Acid Electrophoresis, Staining, Incubation, Concentration Assay

    5) Product Images from "Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin"

    Article Title: Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021075

    In vitro binding and in vivo residence of acetylated avidin. A, Isoelectric focusing of acetylated avidin. Lane 1, IEF standards; Lane 2, Herceptin, streptavidin and ovalbumin mixture; Lane 3, Acetylated avidin 1∶80; Lane 4, Acetylated avidin 1∶40; Lane 5, Acetylated avidin 1∶20. B, Cells were incubated with 150 µM avidin, AvidinOX, acetylated avidin pI 4.7 (Acetylavidin) or oxidized acetylated avidin (OxAcetylavidin) at +4°C and binding revealed by cytofluorimetry with an anti-avidin antibody as detailed in Materials and Methods . Gray peaks are background (second antibody, only). Thin and bold lines, native and oxidized glycoproteins, respectively. C, Avidin and acetylated avidin pI 4.7 (Acetylavidin), were 125 I-labeled and then oxidized with sodium periodate as described in Materials and Methods . All samples were diluted at 3.0 mg/ml in sodium acetate pH 5.5 and 15 µl were injected intramuscularly in one hind limb of mice. After 24 hours, the mice were sacrificed and treated limbs collected, weighed and counted in a gamma counter. Data are expressed as the % of injected dose/100 mg (% ID/100 mg) of tissue. Each point is the average of 5 mice. Bars represent standard deviation. Statistical analysis by Student's t test. ***p
    Figure Legend Snippet: In vitro binding and in vivo residence of acetylated avidin. A, Isoelectric focusing of acetylated avidin. Lane 1, IEF standards; Lane 2, Herceptin, streptavidin and ovalbumin mixture; Lane 3, Acetylated avidin 1∶80; Lane 4, Acetylated avidin 1∶40; Lane 5, Acetylated avidin 1∶20. B, Cells were incubated with 150 µM avidin, AvidinOX, acetylated avidin pI 4.7 (Acetylavidin) or oxidized acetylated avidin (OxAcetylavidin) at +4°C and binding revealed by cytofluorimetry with an anti-avidin antibody as detailed in Materials and Methods . Gray peaks are background (second antibody, only). Thin and bold lines, native and oxidized glycoproteins, respectively. C, Avidin and acetylated avidin pI 4.7 (Acetylavidin), were 125 I-labeled and then oxidized with sodium periodate as described in Materials and Methods . All samples were diluted at 3.0 mg/ml in sodium acetate pH 5.5 and 15 µl were injected intramuscularly in one hind limb of mice. After 24 hours, the mice were sacrificed and treated limbs collected, weighed and counted in a gamma counter. Data are expressed as the % of injected dose/100 mg (% ID/100 mg) of tissue. Each point is the average of 5 mice. Bars represent standard deviation. Statistical analysis by Student's t test. ***p

    Techniques Used: In Vitro, Binding Assay, In Vivo, Avidin-Biotin Assay, Electrofocusing, Incubation, Labeling, Injection, Mouse Assay, Standard Deviation

    In vitro cell binding, in vivo tissue residence and biotin uptake of chemically glycosylated and oxidized streptavidin. A , Binding of streptavidin (SA), low mannose streptavidin (GlySA) and oxidized derivative (OxGlySA) to human prostate carcinoma PC3 and mouse fibroblast NIH-3T3 cells was compared to avidin and AvidinOX. Cells were incubated with 150 µM of either native or oxidized glycoproteins at +4°C and binding revealed by cytofluorimetry with specific primary antibodies as detailed in Materials and Methods . Gray peaks represent background (second antibody, only). Thin and bold lines, native and oxidized glycoproteins, respectively. B , Avidin, streptavidin, low and high mannose streptavidin (corresponding to 26 and 60 % of mannosylated amino groups, respectively) were 125 I-labeled and oxidized with sodium periodate as detailed in Materials and Methods . All samples in their native and oxidized form were diluted to 3.0 mg/ml in sodium acetate pH 5.5 and 15 µl were injected intramuscularly in one hind limb of mice. After 24 hours, the mice were sacrificed and treated limbs collected, muscle weighed and counted in a gamma counter. Data are expressed as the % of injected dose/100 mg (% ID/100 mg) of tissue. Each point is the average of 5 mice. Bars represent standard deviation. Statistical analysis was performed by Student's t test. ***p
    Figure Legend Snippet: In vitro cell binding, in vivo tissue residence and biotin uptake of chemically glycosylated and oxidized streptavidin. A , Binding of streptavidin (SA), low mannose streptavidin (GlySA) and oxidized derivative (OxGlySA) to human prostate carcinoma PC3 and mouse fibroblast NIH-3T3 cells was compared to avidin and AvidinOX. Cells were incubated with 150 µM of either native or oxidized glycoproteins at +4°C and binding revealed by cytofluorimetry with specific primary antibodies as detailed in Materials and Methods . Gray peaks represent background (second antibody, only). Thin and bold lines, native and oxidized glycoproteins, respectively. B , Avidin, streptavidin, low and high mannose streptavidin (corresponding to 26 and 60 % of mannosylated amino groups, respectively) were 125 I-labeled and oxidized with sodium periodate as detailed in Materials and Methods . All samples in their native and oxidized form were diluted to 3.0 mg/ml in sodium acetate pH 5.5 and 15 µl were injected intramuscularly in one hind limb of mice. After 24 hours, the mice were sacrificed and treated limbs collected, muscle weighed and counted in a gamma counter. Data are expressed as the % of injected dose/100 mg (% ID/100 mg) of tissue. Each point is the average of 5 mice. Bars represent standard deviation. Statistical analysis was performed by Student's t test. ***p

    Techniques Used: In Vitro, Binding Assay, In Vivo, Avidin-Biotin Assay, Incubation, Labeling, Injection, Mouse Assay, Standard Deviation

    6) Product Images from "Differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization"

    Article Title: Differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization

    Journal: Open Biology

    doi: 10.1098/rsob.160286

    Chemokines cross-link HS chains dependent on oligomerization; quantitative FRAP analysis. ( a ) CCL5 polymer or its oligomerization mutants, E26A tetramer or E66S dimer (500 nM), ( b ) CXCL11 (500 nM), ( c ) CXCL4 tetramer or its oligomerization mutant, K50E dimer (500 nM), ( d ) CXCL8, CCL2 or CCL7 (500 nM) were incubated on lipid bilayers supporting fluorescently labelled streptavidin to which HS chains were attached to create low-density HS surfaces. ( e ) CCL2 dimer (500 nM), its oligomerization mutant P8A monomer (3 µM), CCL7 (500 nM) or CXCL8 (500 nM) were incubated on lipid bilayers supporting fluorescently labelled streptavidin to which HS chains were attached to create high-density HS surfaces. FRAP was monitored and data fitted to provide (i) the total mobile fraction and (ii) the associated diffusion constant; data are plotted as the mean ± s.e. of two independent experiments ( n = 2). n.s., not significant, * p
    Figure Legend Snippet: Chemokines cross-link HS chains dependent on oligomerization; quantitative FRAP analysis. ( a ) CCL5 polymer or its oligomerization mutants, E26A tetramer or E66S dimer (500 nM), ( b ) CXCL11 (500 nM), ( c ) CXCL4 tetramer or its oligomerization mutant, K50E dimer (500 nM), ( d ) CXCL8, CCL2 or CCL7 (500 nM) were incubated on lipid bilayers supporting fluorescently labelled streptavidin to which HS chains were attached to create low-density HS surfaces. ( e ) CCL2 dimer (500 nM), its oligomerization mutant P8A monomer (3 µM), CCL7 (500 nM) or CXCL8 (500 nM) were incubated on lipid bilayers supporting fluorescently labelled streptavidin to which HS chains were attached to create high-density HS surfaces. FRAP was monitored and data fitted to provide (i) the total mobile fraction and (ii) the associated diffusion constant; data are plotted as the mean ± s.e. of two independent experiments ( n = 2). n.s., not significant, * p

    Techniques Used: Mutagenesis, Incubation, Diffusion-based Assay

    Chemokines cross-link HS chains dependent on oligomerization; representative FRAP micrographs. ( a ) CCL5 or its oligomerization mutants, E26A tetramer or E66S dimer (500 nM), ( b ) CXCL11 (500 nM), ( c ) CXCL4 or its oligomerization mutant, K50E dimer (500 nM) or ( d ) CCL2 (500 nM) or its oligomerization mutant, P8A monomer (3 µM) were incubated on a lipid bilayer supporting fluorescently labelled streptavidin to which HS chains were attached. Following bleaching and recovery analysis, images of the bleached spot were taken to provide visual assessment of the extent of maximal bleaching (0 s, images shown for WT chemokines only) and recovery from bleaching after 300 s (representative of two experiments).
    Figure Legend Snippet: Chemokines cross-link HS chains dependent on oligomerization; representative FRAP micrographs. ( a ) CCL5 or its oligomerization mutants, E26A tetramer or E66S dimer (500 nM), ( b ) CXCL11 (500 nM), ( c ) CXCL4 or its oligomerization mutant, K50E dimer (500 nM) or ( d ) CCL2 (500 nM) or its oligomerization mutant, P8A monomer (3 µM) were incubated on a lipid bilayer supporting fluorescently labelled streptavidin to which HS chains were attached. Following bleaching and recovery analysis, images of the bleached spot were taken to provide visual assessment of the extent of maximal bleaching (0 s, images shown for WT chemokines only) and recovery from bleaching after 300 s (representative of two experiments).

    Techniques Used: Mutagenesis, Incubation

    CXCL4-, CXCL11- and CCL5-mediated modification of high- and low-density HS films. ( a ) CXCL4, ( b ) CXCL11 or ( c ) CCL5 (500 nM) were passed over a QCM-D sensor displaying high-density (high HS) or low-density (low HS) HS films. Alternatively, chemokine was passed over a streptavidin-coated surface with no immobilized HS (no HS) to check non-specific binding (passivation). Subsequent changes in the (i) frequency (a decrease indicates bound chemokine) or (ii) dissipation (a decrease indicates HS film rigidification) as a function of time are plotted. Chemokine injection start and endpoints are indicated by arrows on each curve. The frequency plot for CCL5 demonstrates some non-specific binding (reduction in frequency) to the passivated surface (no HS) that is largely absent with the other chemokines used. This binding is associated with an increase in the dissipation on the HS-free surface, probably as a result of CCL5 polymerization, leading to a slight distortion of the dissipation measurement.
    Figure Legend Snippet: CXCL4-, CXCL11- and CCL5-mediated modification of high- and low-density HS films. ( a ) CXCL4, ( b ) CXCL11 or ( c ) CCL5 (500 nM) were passed over a QCM-D sensor displaying high-density (high HS) or low-density (low HS) HS films. Alternatively, chemokine was passed over a streptavidin-coated surface with no immobilized HS (no HS) to check non-specific binding (passivation). Subsequent changes in the (i) frequency (a decrease indicates bound chemokine) or (ii) dissipation (a decrease indicates HS film rigidification) as a function of time are plotted. Chemokine injection start and endpoints are indicated by arrows on each curve. The frequency plot for CCL5 demonstrates some non-specific binding (reduction in frequency) to the passivated surface (no HS) that is largely absent with the other chemokines used. This binding is associated with an increase in the dissipation on the HS-free surface, probably as a result of CCL5 polymerization, leading to a slight distortion of the dissipation measurement.

    Techniques Used: Modification, QCM-D, Binding Assay, Injection

    CXCL8-, CCL2- and CCL7-mediated modification of high- and low-density HS films. ( a ) CXCL8, ( b ) CCL2 or ( c ) CCL7 (500 nM) were passed over a QCM-D sensor displaying high-density (high HS) or low-density (low HS) HS films. Alternatively, chemokine was passed over a streptavidin-coated surface with no immobilized HS (no HS) to check non-specific binding (passivation). Subsequent changes in the (i) frequency (a decrease indicates bound chemokine) or (ii) dissipation (a decrease indicates HS film rigidification) as a function of time are plotted. Chemokine injection start and endpoints are indicated by arrows on each curve.
    Figure Legend Snippet: CXCL8-, CCL2- and CCL7-mediated modification of high- and low-density HS films. ( a ) CXCL8, ( b ) CCL2 or ( c ) CCL7 (500 nM) were passed over a QCM-D sensor displaying high-density (high HS) or low-density (low HS) HS films. Alternatively, chemokine was passed over a streptavidin-coated surface with no immobilized HS (no HS) to check non-specific binding (passivation). Subsequent changes in the (i) frequency (a decrease indicates bound chemokine) or (ii) dissipation (a decrease indicates HS film rigidification) as a function of time are plotted. Chemokine injection start and endpoints are indicated by arrows on each curve.

    Techniques Used: Modification, QCM-D, Binding Assay, Injection

    Formation of HS films on a passivated QCM-D sensor and for FRAP. Schematic of surfaces used for ( a ) QCM-D and ( b ) FRAP adapted from [ 29 ]. Formation of ( c ) low- and ( d ) high-density HS surfaces on a QCM-D sensor; streptavidin (1 or 20 µg ml −1 ) was flowed over a gold-coated sensor covered with a passivation layer presenting biotin groups until saturation was reached (−24 Hz, no change in dissipation). Biotinylated HS (2 or 5 µg ml −1 ) was then grafted onto this surface to create a low-density HS film (−7 Hz, 1.5 dissipation units) ( c ) or a high-density HS film (−24 Hz, 4.5 dissipation units) ( d ), which could then be used to monitor interactions of HS with chemokines or mutants.
    Figure Legend Snippet: Formation of HS films on a passivated QCM-D sensor and for FRAP. Schematic of surfaces used for ( a ) QCM-D and ( b ) FRAP adapted from [ 29 ]. Formation of ( c ) low- and ( d ) high-density HS surfaces on a QCM-D sensor; streptavidin (1 or 20 µg ml −1 ) was flowed over a gold-coated sensor covered with a passivation layer presenting biotin groups until saturation was reached (−24 Hz, no change in dissipation). Biotinylated HS (2 or 5 µg ml −1 ) was then grafted onto this surface to create a low-density HS film (−7 Hz, 1.5 dissipation units) ( c ) or a high-density HS film (−24 Hz, 4.5 dissipation units) ( d ), which could then be used to monitor interactions of HS with chemokines or mutants.

    Techniques Used: QCM-D

    7) Product Images from "A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy"

    Article Title: A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy

    Journal: Cells

    doi: 10.3390/cells8121510

    Effect of marker nucleotide, the detection approach and dTTP on the signal intensity. The samples containing Lep cells accidentally infected with mycoplasma were fixed by formaldehyde, permeabilized by Triton X-100 and mycoplasmas’ DNA was detected using enzymatic detection with Pol I. ( a ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). ( b ) The enzymatic mixture contained fluorescein-dUTP. ( c ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized by streptavidin conjugated with FITC. ( d ) The enzymatic mixture contained biotin-dUTP and an equimolar amount of dTTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). The images were acquired for 11.44 ms. Scale bar = 10 µm.
    Figure Legend Snippet: Effect of marker nucleotide, the detection approach and dTTP on the signal intensity. The samples containing Lep cells accidentally infected with mycoplasma were fixed by formaldehyde, permeabilized by Triton X-100 and mycoplasmas’ DNA was detected using enzymatic detection with Pol I. ( a ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). ( b ) The enzymatic mixture contained fluorescein-dUTP. ( c ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized by streptavidin conjugated with FITC. ( d ) The enzymatic mixture contained biotin-dUTP and an equimolar amount of dTTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). The images were acquired for 11.44 ms. Scale bar = 10 µm.

    Techniques Used: Marker, Infection, Mass Spectrometry

    8) Product Images from "Quality control for unfolded proteins at the plasma membrane"

    Article Title: Quality control for unfolded proteins at the plasma membrane

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201006012

    Unfolding induced down-regulation of CD4tl-λ C at the PM. (A) Schematic picture of membrane-tethered and soluble bacteriophage λ model proteins. (B) Immunoblot (IB) analysis of CD4tl and CD4tl-λ/λ C expression before (top) or after CHX chase at 37°C for 1.5 h (bottom). Transiently transfected COS7 cells were cultured at 37°C or 26°C before the CHX chase. (C) Indirect immunostaining of thermally unfolded (37°C) or nativelike (26°C) CD4tl-λ C in nonpermeabilized (top) or permeabilized (bottom) COS7 cells. Calreticulin was used as an ER marker. Bars, 10 µm. (D) The PM density of chimeras was determined by ELISA as described in Materials and methods. (E) Pharmacological characterization of rescued and then unfolded CD4tl-λ C degradation as measured by CHX chase and immunoblotting. Lactacystine (LAC), concanamycin (Con), and bafilomycin A1 (Baf) were added simultaneously with CHX for 3.5 h. NH 4 Cl and chloroquin (Chl) were present during the last 2 h of the CHX chase. Equal amounts of proteins were loaded. (F and G) The PM stability of rescued and then unfolded model proteins at 37°C for 1.5 h was determined at 37°C (F) or in the absence of unfolding at 26°C (G) by ELISA. (H) Internalization of unfolded and rescued model proteins was monitored by Ab uptake at 37°C. Data are expressed as the percentage of initial Ab binding. (I) Recycling efficiency was determined by a biotin-streptavidin sandwich assay as described in Materials and methods and expressed as the percentage of CD4tl. Molecular mass is given in kilodaltons. The data shown represent means ± SEM.
    Figure Legend Snippet: Unfolding induced down-regulation of CD4tl-λ C at the PM. (A) Schematic picture of membrane-tethered and soluble bacteriophage λ model proteins. (B) Immunoblot (IB) analysis of CD4tl and CD4tl-λ/λ C expression before (top) or after CHX chase at 37°C for 1.5 h (bottom). Transiently transfected COS7 cells were cultured at 37°C or 26°C before the CHX chase. (C) Indirect immunostaining of thermally unfolded (37°C) or nativelike (26°C) CD4tl-λ C in nonpermeabilized (top) or permeabilized (bottom) COS7 cells. Calreticulin was used as an ER marker. Bars, 10 µm. (D) The PM density of chimeras was determined by ELISA as described in Materials and methods. (E) Pharmacological characterization of rescued and then unfolded CD4tl-λ C degradation as measured by CHX chase and immunoblotting. Lactacystine (LAC), concanamycin (Con), and bafilomycin A1 (Baf) were added simultaneously with CHX for 3.5 h. NH 4 Cl and chloroquin (Chl) were present during the last 2 h of the CHX chase. Equal amounts of proteins were loaded. (F and G) The PM stability of rescued and then unfolded model proteins at 37°C for 1.5 h was determined at 37°C (F) or in the absence of unfolding at 26°C (G) by ELISA. (H) Internalization of unfolded and rescued model proteins was monitored by Ab uptake at 37°C. Data are expressed as the percentage of initial Ab binding. (I) Recycling efficiency was determined by a biotin-streptavidin sandwich assay as described in Materials and methods and expressed as the percentage of CD4tl. Molecular mass is given in kilodaltons. The data shown represent means ± SEM.

    Techniques Used: Expressing, Transfection, Cell Culture, Immunostaining, Marker, Enzyme-linked Immunosorbent Assay, Binding Assay

    9) Product Images from "pH-Dependent Deformations of the Energy Landscape of Avidin-like Proteins Investigated by Single Molecule Force Spectroscopy"

    Article Title: pH-Dependent Deformations of the Energy Landscape of Avidin-like Proteins Investigated by Single Molecule Force Spectroscopy

    Journal: Molecules

    doi: 10.3390/molecules190812531

    ( a ) Tip and support chemistry. Upper part: APTES functionalized AFM tips are reacted with the heterobifunctional PEG tether NHS-PEG-biotin resulting in a covalent amide bond formation. Lower part: avidin (or avidin-like protein) is covalently bound to APTES coated mica using a homo-bifunctional EGS crosslinker. ( b ) Typical force distance cycle. The distance dependent cantilever bending is shown in red for the approaching period and in black for the retraction. In the latter a typical unbinding event, visible as parabolic shaped downwards bending can be seen (highlighted with an arrow). In contrast, the inset represents a force distance cycle without any specific interaction. ( c ) Specificity proof exemplary shown for streptavidin at a pulling velocity of 400 nm/s at pH 7. The black line represents the probability density function using a biotin tethered tip, whereas the red line shows the probability density function of the very same system after blocking the tip by adding free streptavidin.
    Figure Legend Snippet: ( a ) Tip and support chemistry. Upper part: APTES functionalized AFM tips are reacted with the heterobifunctional PEG tether NHS-PEG-biotin resulting in a covalent amide bond formation. Lower part: avidin (or avidin-like protein) is covalently bound to APTES coated mica using a homo-bifunctional EGS crosslinker. ( b ) Typical force distance cycle. The distance dependent cantilever bending is shown in red for the approaching period and in black for the retraction. In the latter a typical unbinding event, visible as parabolic shaped downwards bending can be seen (highlighted with an arrow). In contrast, the inset represents a force distance cycle without any specific interaction. ( c ) Specificity proof exemplary shown for streptavidin at a pulling velocity of 400 nm/s at pH 7. The black line represents the probability density function using a biotin tethered tip, whereas the red line shows the probability density function of the very same system after blocking the tip by adding free streptavidin.

    Techniques Used: Avidin-Biotin Assay, Blocking Assay

    ( a ) Comparison of the binding probabilities of D-biotin with avidin (gray), chimeric avidin (red), and streptavidin (blue) at different pH values. ( b ) Complex lifetimes τ of avidin-biotin (gray) and chimeric avidin-biotin (red) at different pH values. ( c ) Loading rate dependence of the most probable unbinding force of avidin (left) and chimeric avidin (right) at pH values ranging from 1–11.
    Figure Legend Snippet: ( a ) Comparison of the binding probabilities of D-biotin with avidin (gray), chimeric avidin (red), and streptavidin (blue) at different pH values. ( b ) Complex lifetimes τ of avidin-biotin (gray) and chimeric avidin-biotin (red) at different pH values. ( c ) Loading rate dependence of the most probable unbinding force of avidin (left) and chimeric avidin (right) at pH values ranging from 1–11.

    Techniques Used: Binding Assay, Avidin-Biotin Assay

    ( a ) Molecular structure of D-biotin in the β-barrel avidin (PDB 2AVI). ( d ) AFM image of a covalently bound avidin layer. Scratching of a 0.5x0.5 µm area results in a hole. In the cross-section below (corresponding to the yellow line in the AFM image) the height difference of 2.07 ± 0.47 nm can be seen between avidin and the support. ( b ) Molecular structure of D-biotin in the β-barrel chimeric avidin prepared by positioning the ligand to the apo structure of chimeric avidin (PDB 3MMO) by using avidin-biotin complex as a template. ( e ) AFM image of a covalently bound chimeric avidin layer. Image size is 2.5 × 2.5 µm, z-scale bar is 7 nm. The height difference in the cross-sections is 1.86 ± 0.26 nm. ( c ) Molecular structure of D-biotin in the β-barrel streptavidin (PDB 1MK5). ( f ) AFM image of a covalently bound streptavidin layer. Image size is 2.5 × 2.5 µm, x-scale bar is 1 µm. The height difference in the cross-sections is 2.03 ± 0.15 nm. The molecular representations were prepared by using the program VMD.
    Figure Legend Snippet: ( a ) Molecular structure of D-biotin in the β-barrel avidin (PDB 2AVI). ( d ) AFM image of a covalently bound avidin layer. Scratching of a 0.5x0.5 µm area results in a hole. In the cross-section below (corresponding to the yellow line in the AFM image) the height difference of 2.07 ± 0.47 nm can be seen between avidin and the support. ( b ) Molecular structure of D-biotin in the β-barrel chimeric avidin prepared by positioning the ligand to the apo structure of chimeric avidin (PDB 3MMO) by using avidin-biotin complex as a template. ( e ) AFM image of a covalently bound chimeric avidin layer. Image size is 2.5 × 2.5 µm, z-scale bar is 7 nm. The height difference in the cross-sections is 1.86 ± 0.26 nm. ( c ) Molecular structure of D-biotin in the β-barrel streptavidin (PDB 1MK5). ( f ) AFM image of a covalently bound streptavidin layer. Image size is 2.5 × 2.5 µm, x-scale bar is 1 µm. The height difference in the cross-sections is 2.03 ± 0.15 nm. The molecular representations were prepared by using the program VMD.

    Techniques Used: Avidin-Biotin Assay

    10) Product Images from "Atomic Force Microscope Imaging of Chromatin Assembled in Xenopus laevis Egg Extract"

    Article Title: Atomic Force Microscope Imaging of Chromatin Assembled in Xenopus laevis Egg Extract

    Journal: Chromosoma

    doi: 10.1007/s00412-010-0307-4

    Direct Imaging Method for AFM of Higher-Order Structures. AFM images of chromatin assembled from lambda phage DNA in Xenopus laevis egg extract on streptavidin-coated surface and imaged directly on the same surface. (a) The control surface without DNA
    Figure Legend Snippet: Direct Imaging Method for AFM of Higher-Order Structures. AFM images of chromatin assembled from lambda phage DNA in Xenopus laevis egg extract on streptavidin-coated surface and imaged directly on the same surface. (a) The control surface without DNA

    Techniques Used: Imaging

    11) Product Images from "Electrolyte-free Amperometric Immunosensor using a Dendritic Nanotip"

    Article Title: Electrolyte-free Amperometric Immunosensor using a Dendritic Nanotip

    Journal: RSC advances

    doi: 10.1039/C3RA40262B

    (a) Cyclic voltammetry for a dendritic nanotip made of Si nanowires and SWCNTs. The sweeping rates vary from 100 mV/sec to 1 V/sec (b) I–V response for a dendritic nanotip without coating, with coating of streptavidin, and streptavidin + biotinylated
    Figure Legend Snippet: (a) Cyclic voltammetry for a dendritic nanotip made of Si nanowires and SWCNTs. The sweeping rates vary from 100 mV/sec to 1 V/sec (b) I–V response for a dendritic nanotip without coating, with coating of streptavidin, and streptavidin + biotinylated

    Techniques Used: Size-exclusion Chromatography

    12) Product Images from "Liposomes on a streptavidin crystal: a system to study membrane proteins by cryo-EM"

    Article Title: Liposomes on a streptavidin crystal: a system to study membrane proteins by cryo-EM

    Journal: Methods in enzymology

    doi: 10.1016/S0076-6879(10)81007-9

    The streptavidin crystal as a tethering substrate. (A) Side view of the streptavidin tetramer. One bound biotin is shown as a space-filling model, and the other three binding sites are marked with asterisks. (B) 2D crystals are formed by attaching streptavidin molecules to a lipid monolayer via specific biotin-avidin binding. Biotinylated lipid molecules are indicated by triangles. (C-D) Negative-stain EM images show crystals grown from 0.2 mg/ml and 0.05 mg/ml streptavidin solutions for 2 hours at room temperature. The black lines outline the domain boundaries, the arrow shows the crystal orientation in each region, and the insets are 3× close-ups. (E) Dependence of the average domain size on the protein concentration and growth time. The crystal growth time was varied from 2 hr (dashed line) to 6 hr (solid line) and overnight (data not shown, but the crystal covered entire 2 μm-diameter holes for 0.2 mg/ml of protein concentration). Error bars are the standard deviation of crystal domain sizes with n = 20. (F) The dependence of the average liposome density on the incubation time. The lines represent the liposome densities on 2D crystal (solid line) and on a continuous carbon support film (dashed line), respectively.
    Figure Legend Snippet: The streptavidin crystal as a tethering substrate. (A) Side view of the streptavidin tetramer. One bound biotin is shown as a space-filling model, and the other three binding sites are marked with asterisks. (B) 2D crystals are formed by attaching streptavidin molecules to a lipid monolayer via specific biotin-avidin binding. Biotinylated lipid molecules are indicated by triangles. (C-D) Negative-stain EM images show crystals grown from 0.2 mg/ml and 0.05 mg/ml streptavidin solutions for 2 hours at room temperature. The black lines outline the domain boundaries, the arrow shows the crystal orientation in each region, and the insets are 3× close-ups. (E) Dependence of the average domain size on the protein concentration and growth time. The crystal growth time was varied from 2 hr (dashed line) to 6 hr (solid line) and overnight (data not shown, but the crystal covered entire 2 μm-diameter holes for 0.2 mg/ml of protein concentration). Error bars are the standard deviation of crystal domain sizes with n = 20. (F) The dependence of the average liposome density on the incubation time. The lines represent the liposome densities on 2D crystal (solid line) and on a continuous carbon support film (dashed line), respectively.

    Techniques Used: Binding Assay, Avidin-Biotin Assay, Staining, Protein Concentration, Standard Deviation, Incubation

    Liposomes on a streptavidin crystal. (A) Scheme of the tethering of liposomes in a vitreous ice layer (light gray) on a 2D crystal substrate (gray) spanning a hole in the carbon support film (black). (B) Scale drawing of the tethering system. The 2D crystal layer (5.0 nm thick) is bound to a lipid monolayer (2.5 nm) which spans a hole in the perforated carbon film (15–30 nm in thickness). (C) Portion of a cryo-EM micrograph showing proteoliposomes and the streptavidin-crystal background. (D) The same image, after the contribution of lipid membrane and 2D crystals is removed. Manually-picked particles are marked by white boxes.
    Figure Legend Snippet: Liposomes on a streptavidin crystal. (A) Scheme of the tethering of liposomes in a vitreous ice layer (light gray) on a 2D crystal substrate (gray) spanning a hole in the carbon support film (black). (B) Scale drawing of the tethering system. The 2D crystal layer (5.0 nm thick) is bound to a lipid monolayer (2.5 nm) which spans a hole in the perforated carbon film (15–30 nm in thickness). (C) Portion of a cryo-EM micrograph showing proteoliposomes and the streptavidin-crystal background. (D) The same image, after the contribution of lipid membrane and 2D crystals is removed. Manually-picked particles are marked by white boxes.

    Techniques Used:

    13) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.355107

    Degradins induce ER to cytosol retrotranslocation. Western blot of cellular extracts from HEK293 cells co-transfected with vectors expressing the target protein mdα and the indicated degradins ( a and b ), or from stable transfectants HEK-mdα and clone G6 ( c ) in the presence or absence of the proteasome inhibitor MG132, as indicated. Blots were developed with anti-FcϵRI. d and e , Western blot retardation assay performed on extracts derived from cells co-transfected with the BAP-containing targets, sα-BAP ( d ) and BAP-mdα ( e ), the indicated degradins and cytosolic BirA, treated or not treated with MG132. Samples were run in the presence or absence of streptavidin ( StrAv ), as indicated. Biotinylated (retrotranslocated) molecules have a retarded PAGE mobility, indicated by arrows . Blots were developed with anti-SV5. Degradins used did not contain the SV5 tag, which was instead present in the targets.
    Figure Legend Snippet: Degradins induce ER to cytosol retrotranslocation. Western blot of cellular extracts from HEK293 cells co-transfected with vectors expressing the target protein mdα and the indicated degradins ( a and b ), or from stable transfectants HEK-mdα and clone G6 ( c ) in the presence or absence of the proteasome inhibitor MG132, as indicated. Blots were developed with anti-FcϵRI. d and e , Western blot retardation assay performed on extracts derived from cells co-transfected with the BAP-containing targets, sα-BAP ( d ) and BAP-mdα ( e ), the indicated degradins and cytosolic BirA, treated or not treated with MG132. Samples were run in the presence or absence of streptavidin ( StrAv ), as indicated. Biotinylated (retrotranslocated) molecules have a retarded PAGE mobility, indicated by arrows . Blots were developed with anti-SV5. Degradins used did not contain the SV5 tag, which was instead present in the targets.

    Techniques Used: Western Blot, Transfection, Expressing, Derivative Assay, Polyacrylamide Gel Electrophoresis

    Degradins decay with the target. a , [ 35 S]methionine pulse-chase labeling of cells co-transfected with L-dg and the irrelevant (MHC-Iα) or specific (mdα) targets. L-dg ( arrow ) was immunoprecipitated at different chase time points, as indicated. The band intensity was determined relative to the 0 min time point. b , a similar experiment as described in a showing immunoprecipitates of mock (MHC-Iα, open arrow ) or specific (mdα, solid arrow ) targets. Immunoprecipitates were resolved by PAGE and revealed by autoradiography. c , Western blot retardation assay on extracts derived from cells co-transfected with the BAP-tagged L-dg, cytosolic BirA, and mock (MHC-Iα) or specific (mdα) targets, treated or not treated with MG132. Samples were run in the presence or absence of streptavidin ( StrAv ), as indicated. Arrowhead indicates biotinylated (retrotranslocated) molecules. Blot was developed with anti-SV5 and anti-actin.
    Figure Legend Snippet: Degradins decay with the target. a , [ 35 S]methionine pulse-chase labeling of cells co-transfected with L-dg and the irrelevant (MHC-Iα) or specific (mdα) targets. L-dg ( arrow ) was immunoprecipitated at different chase time points, as indicated. The band intensity was determined relative to the 0 min time point. b , a similar experiment as described in a showing immunoprecipitates of mock (MHC-Iα, open arrow ) or specific (mdα, solid arrow ) targets. Immunoprecipitates were resolved by PAGE and revealed by autoradiography. c , Western blot retardation assay on extracts derived from cells co-transfected with the BAP-tagged L-dg, cytosolic BirA, and mock (MHC-Iα) or specific (mdα) targets, treated or not treated with MG132. Samples were run in the presence or absence of streptavidin ( StrAv ), as indicated. Arrowhead indicates biotinylated (retrotranslocated) molecules. Blot was developed with anti-SV5 and anti-actin.

    Techniques Used: Pulse Chase, Labeling, Transfection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Western Blot, Derivative Assay

    14) Product Images from "Small molecule detection with aptamer based lateral flow assays: Applying aptamer-C-reactive protein cross-recognition for ampicillin detection"

    Article Title: Small molecule detection with aptamer based lateral flow assays: Applying aptamer-C-reactive protein cross-recognition for ampicillin detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23963-6

    Schematic illustration of the designed competitive LFA for ampicillin detection. Aptamer-mFc-AuNP conjugates are incubated with biotinylated CRP in the presence or absence of ampicillin. If ampicillin is absent, the conjugates will bind towards biotinylated CRP and will be immobilized on the test line with streptavidin ( A ). If ampicillin is present, CRP is displaced from the aptamer, leading to a signal decrease on the test line ( B ). In both cases, the mFc on the conjugates leads to an immobilization on the control line via the α-mouse antibody, generating the control line.
    Figure Legend Snippet: Schematic illustration of the designed competitive LFA for ampicillin detection. Aptamer-mFc-AuNP conjugates are incubated with biotinylated CRP in the presence or absence of ampicillin. If ampicillin is absent, the conjugates will bind towards biotinylated CRP and will be immobilized on the test line with streptavidin ( A ). If ampicillin is present, CRP is displaced from the aptamer, leading to a signal decrease on the test line ( B ). In both cases, the mFc on the conjugates leads to an immobilization on the control line via the α-mouse antibody, generating the control line.

    Techniques Used: Incubation

    15) Product Images from "Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction"

    Article Title: Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S76192

    Process of molecular translator and ISDPR. Notes: ( A ) Principle of molecular translator, the input GDNF is converted into output DNA3 by the binding-induced strand-displacement activity of the molecular translator. ( B ) The output DNA3 triggers ISDPR for signal amplification by producing a large amount of duplex DNA. ( C ) Schematic illustration of the strip biosensor for visual detection of duplex DNA. AuNP–antidigoxin antibody is dispensed on the conjugate pad. Streptavidin and secondary antibody are dispensed on the test zone and control zone, respectively. Abbreviations: ISDPR, isothermal strand-displacement polymerization reaction; GDNF, glial cell line-derived neurotrophic factor; DNA, deoxyribonucleic acid; AuNP, gold nanoparticle; NC, nitrocellulose.
    Figure Legend Snippet: Process of molecular translator and ISDPR. Notes: ( A ) Principle of molecular translator, the input GDNF is converted into output DNA3 by the binding-induced strand-displacement activity of the molecular translator. ( B ) The output DNA3 triggers ISDPR for signal amplification by producing a large amount of duplex DNA. ( C ) Schematic illustration of the strip biosensor for visual detection of duplex DNA. AuNP–antidigoxin antibody is dispensed on the conjugate pad. Streptavidin and secondary antibody are dispensed on the test zone and control zone, respectively. Abbreviations: ISDPR, isothermal strand-displacement polymerization reaction; GDNF, glial cell line-derived neurotrophic factor; DNA, deoxyribonucleic acid; AuNP, gold nanoparticle; NC, nitrocellulose.

    Techniques Used: Binding Assay, Activity Assay, Amplification, Stripping Membranes, Derivative Assay

    16) Product Images from "The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes"

    Article Title: The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes

    Journal: The Journal of Cell Biology

    doi:

    Identification of surface proteins by vectorial labeling with biotin. ( A ) Activated mt+ gametes were labeled with Sulfo-NHS-Biotin, fertilization tubules were isolated, and samples (12 μg) of homogenized cells (lane HC ), sucrose pellets (lane SP ), and Percoll pellets (lane PP ) were separated by SDS-PAGE and analyzed by streptavidin blotting. Arrows indicate the locations of surface-biotinylated proteins of 500 ( ft500 ) and 350 kD ( ft350 ). ( B ) Staining with Coomassie blue of the same samples as in A. Arrows indicate the locations of ft500 and ft350. ( C ) To identify proteins biotinylated in disrupted cells, activated mt+ gametes were homogenized before biotinylation (lane H → B ) and 1.5 μg of protein was analyzed by SDS-PAGE and streptavidin blotting. In addition, potential streptavidin-binding proteins were identified by analyzing both nonbiotinylated homogenized cells (12 μg, HC , − B ) and fertilization tubules purified from nonbiotinylated cells (12 μg, PP , − B ).
    Figure Legend Snippet: Identification of surface proteins by vectorial labeling with biotin. ( A ) Activated mt+ gametes were labeled with Sulfo-NHS-Biotin, fertilization tubules were isolated, and samples (12 μg) of homogenized cells (lane HC ), sucrose pellets (lane SP ), and Percoll pellets (lane PP ) were separated by SDS-PAGE and analyzed by streptavidin blotting. Arrows indicate the locations of surface-biotinylated proteins of 500 ( ft500 ) and 350 kD ( ft350 ). ( B ) Staining with Coomassie blue of the same samples as in A. Arrows indicate the locations of ft500 and ft350. ( C ) To identify proteins biotinylated in disrupted cells, activated mt+ gametes were homogenized before biotinylation (lane H → B ) and 1.5 μg of protein was analyzed by SDS-PAGE and streptavidin blotting. In addition, potential streptavidin-binding proteins were identified by analyzing both nonbiotinylated homogenized cells (12 μg, HC , − B ) and fertilization tubules purified from nonbiotinylated cells (12 μg, PP , − B ).

    Techniques Used: Labeling, Isolation, SDS Page, Staining, Binding Assay, Purification

    17) Product Images from "Single-Molecule Analysis Reveals the Kinetics and Physiological Relevance of MutL-ssDNA Binding"

    Article Title: Single-Molecule Analysis Reveals the Kinetics and Physiological Relevance of MutL-ssDNA Binding

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015496

    Single-molecule FRET analysis of MutL-ssDNA binding. ( A ) Schematic representation of a single-molecule FRET system. The Evanescent field resulting from Total Internal Reflection excites a Cy3 donor fluorophore that may FRET with a Cy5 acceptor fluorophore. A partial duplex DNA attached to the PEG-biotin surface via a biotin-streptavidin linker contains a 5′-dT33 single-stranded tail labeled with the donor Cy3 and an acceptor Cy5 linked to the dsDNA/ssDNA junction. The distance between fluorophores changes with MutL-ssDNA binding affecting the FRET efficiency. ( B ) Representative single molecule trace of the Cy3 and Cy5 fluorescence intensity (top panel) and the resulting FRET (bottom panel) with 50 nM MutL in 25 mM NaCl. The binding of MutL reduces the FRET. ( C ) A transition density plot in the presence of MutL. There are two FRET states of 0.44 and 0.26 FRET values, respectively. The plot consists of 2,362 transitions from 257 traces. ( D ) Histogram of FRET values from populations of single molecules normalized to the peak count. The FRET efficiency in the absence of MutL is 0.41±0.15 (mean ± s.d.; n = 74,634 points). ( E ) Representative trace in the presence of 50 nM MutL in 25 mM NaCl. The FRET efficiency for τ on and τ off represent the association and dissociation time of MutL, respectively (upper panel). Rate constants, k on = 1/ τ off and k off = 1/ τ on were determined by fitting an exponential decay function to the histogram derived from a population of dwell times (lower panels).
    Figure Legend Snippet: Single-molecule FRET analysis of MutL-ssDNA binding. ( A ) Schematic representation of a single-molecule FRET system. The Evanescent field resulting from Total Internal Reflection excites a Cy3 donor fluorophore that may FRET with a Cy5 acceptor fluorophore. A partial duplex DNA attached to the PEG-biotin surface via a biotin-streptavidin linker contains a 5′-dT33 single-stranded tail labeled with the donor Cy3 and an acceptor Cy5 linked to the dsDNA/ssDNA junction. The distance between fluorophores changes with MutL-ssDNA binding affecting the FRET efficiency. ( B ) Representative single molecule trace of the Cy3 and Cy5 fluorescence intensity (top panel) and the resulting FRET (bottom panel) with 50 nM MutL in 25 mM NaCl. The binding of MutL reduces the FRET. ( C ) A transition density plot in the presence of MutL. There are two FRET states of 0.44 and 0.26 FRET values, respectively. The plot consists of 2,362 transitions from 257 traces. ( D ) Histogram of FRET values from populations of single molecules normalized to the peak count. The FRET efficiency in the absence of MutL is 0.41±0.15 (mean ± s.d.; n = 74,634 points). ( E ) Representative trace in the presence of 50 nM MutL in 25 mM NaCl. The FRET efficiency for τ on and τ off represent the association and dissociation time of MutL, respectively (upper panel). Rate constants, k on = 1/ τ off and k off = 1/ τ on were determined by fitting an exponential decay function to the histogram derived from a population of dwell times (lower panels).

    Techniques Used: Binding Assay, Labeling, Fluorescence, Derivative Assay

    18) Product Images from "Unique surface sensing property and enhanced sensitivity in microring resonator biosensors based on subwavelength grating waveguides"

    Article Title: Unique surface sensing property and enhanced sensitivity in microring resonator biosensors based on subwavelength grating waveguides

    Journal: Optics Express

    doi: 10.1364/OE.24.029724

    (a) Real time monitoring of the resonance shift in SWG microring biosensor during the biosensing experiment; Blue region indicate buffer washing steps and other steps are marked with the corresponding reagents used. Anti-SA: anti-streptavidin antibody, SA: streptavidin, bio-BSA: biotinylated BSA. (b) Resonance shift in both SWG microring and regular microring; insets show SEM images of both microrings; GLU: glutaraldehyde (c) Surface sensitivity with respect to estimated thickness in both SWG microring and regular microring.
    Figure Legend Snippet: (a) Real time monitoring of the resonance shift in SWG microring biosensor during the biosensing experiment; Blue region indicate buffer washing steps and other steps are marked with the corresponding reagents used. Anti-SA: anti-streptavidin antibody, SA: streptavidin, bio-BSA: biotinylated BSA. (b) Resonance shift in both SWG microring and regular microring; insets show SEM images of both microrings; GLU: glutaraldehyde (c) Surface sensitivity with respect to estimated thickness in both SWG microring and regular microring.

    Techniques Used:

    19) Product Images from "Electrochemical Immunosensor Detection of Urinary Lactoferrin in Clinical Samples for Urinary Tract Infection Diagnosis"

    Article Title: Electrochemical Immunosensor Detection of Urinary Lactoferrin in Clinical Samples for Urinary Tract Infection Diagnosis

    Journal: Biosensors & bioelectronics

    doi: 10.1016/j.bios.2010.07.002

    (A) Schematic of the electrochemical immunosensor assay, with sandwich detection of the analyte by a biotinylated capture antibody and a HRP-conjugated detector antibody. (B) Comparison of the binding affinity of 3 different LTF capture antibodies directly conjugated to the SAM: a monoclonal Ab (mAb), a polyclonal Ab (pAb), and a biotinylated polyclonal Ab (biotin-pAb). The biotin-pAb has the best signal-to-noise (S/N) ratio for electrochemical detection of LTF. (C) Comparison of capture antibody immobilization techniques through direct covalent binding to the SAM (2, red) or indirect binding thorough a biotin-streptavidin-biotin linkage (4, light blue). Non-specific signals for both immobilization strategies were measured by substituting capture antibody with BSA (1, dark blue and 3, yellow). Each bar represents the mean value from duplicate experiments.
    Figure Legend Snippet: (A) Schematic of the electrochemical immunosensor assay, with sandwich detection of the analyte by a biotinylated capture antibody and a HRP-conjugated detector antibody. (B) Comparison of the binding affinity of 3 different LTF capture antibodies directly conjugated to the SAM: a monoclonal Ab (mAb), a polyclonal Ab (pAb), and a biotinylated polyclonal Ab (biotin-pAb). The biotin-pAb has the best signal-to-noise (S/N) ratio for electrochemical detection of LTF. (C) Comparison of capture antibody immobilization techniques through direct covalent binding to the SAM (2, red) or indirect binding thorough a biotin-streptavidin-biotin linkage (4, light blue). Non-specific signals for both immobilization strategies were measured by substituting capture antibody with BSA (1, dark blue and 3, yellow). Each bar represents the mean value from duplicate experiments.

    Techniques Used: Binding Assay

    20) Product Images from "Antibodies to a strain-specific citrullinated Epstein-Barr virus peptide diagnoses rheumatoid arthritis"

    Article Title: Antibodies to a strain-specific citrullinated Epstein-Barr virus peptide diagnoses rheumatoid arthritis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22058-6

    Reactivity of rheumatoid arthritis sera and healthy donor sera to linear and cyclic EBNA-2 peptides and control peptides analysed by streptavidin capture ELISA. ( a ) Reactivity of rheumatoid arthritis sera (n = 10) to cyclic and linear N/C-terminally biotinylated EBNA-2 peptides (amino acids 313–333 of Epstein-Barr virus strain AG876). “B” represents the location of the biotin labeling in relation the “EBNA” peptide. L = linear, C = cyclic. Linear peptide: GQGRGRWRG-Cit-GRSKGRGRMH-B, cyclic peptide: GQGRCGRWRG-Cit-GRSKGRGCRMH-B. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to healthy controls was used for comparison. ( b ) Reactivity of healthy donor sera (n = 10) to linear N/C-terminally biotinylated EBNA-2 peptides. ( c ) Reactivity of rheumatoid arthritis sera (n = 20) to linear and cyclic EBNA-2 peptide linked to a C-terminal biotin (amino acids 313–333 of Epstein-Barr virus strain AG876). Non-citrullinated peptides (Arg) were used at controls. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to non-citrullinated peptides is used for comparison. ( d ) Reactivity of healthy donor sera to linear and cyclic EBNA-2 peptides linked to a C-terminal biotin (n = 20).
    Figure Legend Snippet: Reactivity of rheumatoid arthritis sera and healthy donor sera to linear and cyclic EBNA-2 peptides and control peptides analysed by streptavidin capture ELISA. ( a ) Reactivity of rheumatoid arthritis sera (n = 10) to cyclic and linear N/C-terminally biotinylated EBNA-2 peptides (amino acids 313–333 of Epstein-Barr virus strain AG876). “B” represents the location of the biotin labeling in relation the “EBNA” peptide. L = linear, C = cyclic. Linear peptide: GQGRGRWRG-Cit-GRSKGRGRMH-B, cyclic peptide: GQGRCGRWRG-Cit-GRSKGRGCRMH-B. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to healthy controls was used for comparison. ( b ) Reactivity of healthy donor sera (n = 10) to linear N/C-terminally biotinylated EBNA-2 peptides. ( c ) Reactivity of rheumatoid arthritis sera (n = 20) to linear and cyclic EBNA-2 peptide linked to a C-terminal biotin (amino acids 313–333 of Epstein-Barr virus strain AG876). Non-citrullinated peptides (Arg) were used at controls. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to non-citrullinated peptides is used for comparison. ( d ) Reactivity of healthy donor sera to linear and cyclic EBNA-2 peptides linked to a C-terminal biotin (n = 20).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Labeling

    Reactivity of rheumatoid arthritis sera and healthy donor sera to substituted and truncated linear EBNA-2 peptides analysed by streptavidin capture ELISA. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to healthy controls is used for comparison. ( a ) Reactivity of rheumatoid arthritis sera to EBNA-2 peptides (n = 10). ( b ) Reactivity of healthy donor sera to EBNA-2 peptides (n = 10).
    Figure Legend Snippet: Reactivity of rheumatoid arthritis sera and healthy donor sera to substituted and truncated linear EBNA-2 peptides analysed by streptavidin capture ELISA. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to healthy controls is used for comparison. ( a ) Reactivity of rheumatoid arthritis sera to EBNA-2 peptides (n = 10). ( b ) Reactivity of healthy donor sera to EBNA-2 peptides (n = 10).

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Reactivity of rheumatoid arthritis and healthy donor sera to selected linear EBNA-2 peptides originating from three Epstein-Barr virus strains anslysed by streptavidin capture ELISA. *Specificity is calculated based on the reactivity of 10 healthy donor sera (HD) to the specific peptides. Statistical calculations are performed using the Student’s t-test, where antibody reactivity to healthy controls is used for comparison. a. Reactivity of rheumatoid arthritis sera (n = 15). b. Reactivity of healthy donor sera (n = 10).
    Figure Legend Snippet: Reactivity of rheumatoid arthritis and healthy donor sera to selected linear EBNA-2 peptides originating from three Epstein-Barr virus strains anslysed by streptavidin capture ELISA. *Specificity is calculated based on the reactivity of 10 healthy donor sera (HD) to the specific peptides. Statistical calculations are performed using the Student’s t-test, where antibody reactivity to healthy controls is used for comparison. a. Reactivity of rheumatoid arthritis sera (n = 15). b. Reactivity of healthy donor sera (n = 10).

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Reactivity of rheumatoid arthritis sera and control sera to the linear EBNA-2 peptide analysed by streptavidin capture ELISA and in the commercial CCPlus and CCP3.1 assays. The following sera were selected for analysis; RA (n = 126), HD (n = 80), SLE (n = 20) and SjS (n = 40). ( a ) Reactivity of RA sera and control sera in the CCP3.1 assay. ( b ) Reactivity of RA sera and control sera in the CCPlus assay. ( c ) Reactivity of RA sera and control sera to the linear EBNA-2 peptide.
    Figure Legend Snippet: Reactivity of rheumatoid arthritis sera and control sera to the linear EBNA-2 peptide analysed by streptavidin capture ELISA and in the commercial CCPlus and CCP3.1 assays. The following sera were selected for analysis; RA (n = 126), HD (n = 80), SLE (n = 20) and SjS (n = 40). ( a ) Reactivity of RA sera and control sera in the CCP3.1 assay. ( b ) Reactivity of RA sera and control sera in the CCPlus assay. ( c ) Reactivity of RA sera and control sera to the linear EBNA-2 peptide.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    21) Product Images from "Surface-Anchored Monomeric Agonist pMHCs Alone Trigger TCR with High Sensitivity "

    Article Title: Surface-Anchored Monomeric Agonist pMHCs Alone Trigger TCR with High Sensitivity

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0060043

    T Cells Respond to Agonist pMHC in a Sublinear Fashion AD10 T cell calcium responses to IEk-MCC diluted by IEk-HSP70 on lipid bilayers in Figure 2 B and AD10 T cell IL2 responses to bio-IEk-MCC on streptavidin plates in Figure 3 A are plotted as power functions of the calculated number of IEk-MCC per cell. T cell responses are shown as the ratio of responding to nonresponding cells. Also shown are theoretical curves of linear and second-degree function responses.
    Figure Legend Snippet: T Cells Respond to Agonist pMHC in a Sublinear Fashion AD10 T cell calcium responses to IEk-MCC diluted by IEk-HSP70 on lipid bilayers in Figure 2 B and AD10 T cell IL2 responses to bio-IEk-MCC on streptavidin plates in Figure 3 A are plotted as power functions of the calculated number of IEk-MCC per cell. T cell responses are shown as the ratio of responding to nonresponding cells. Also shown are theoretical curves of linear and second-degree function responses.

    Techniques Used:

    Very Low Numbers of Agonist IEk Anchored on a Fixed Surface Induce AD10 T Cell IL2 Production, Independent of Endogenous or Null IEk, or IAk (A) Bio-IEk-MCC, bio-IEk-ER60, bio-IEk-HSP70, or bio-IEk-99A at the indicated concentrations were added to 96-well plates precoated with streptavidin and incubated for 18 h at 37 °C. AD10 CD4 + T cells were added to the wells and stimulated for 7 h prior to IL2 assay. (B) The level of IEk proteins on streptavidin plates coated as in (A) was determined by ELISA using anti-IEk 14-4-4s antibody followed by HRP-conjugated goat anti-mouse antibody. ABTS substrate color development was monitored at 405 nm. (C) Streptavidin plates coated with bio-IEk-MCC at the indicated concentrations were subsequently saturated with 10 μg/ml bio-IEk-ER60, bio-IEk-HSP70, bio-IEk-99A, or buffer for 1 h at room temperature before being used to stimulate AD10 T cells. (D) The 96-well ELISA plates were coated with bio-IEk-MCC at the indicated concentrations overnight at 4 °C. The wells were then saturated with 10 μg/ml BSA, IEk-ER60, IEk-HSP70, IEk-99A, or IAk-CA for 4 h at room temperature. Stimulation of AD10 and measurement of IL2 measurements were done as described in (A). (E and F) Same as (C) and (D), respectively, except that 5C.C7 cells were used.
    Figure Legend Snippet: Very Low Numbers of Agonist IEk Anchored on a Fixed Surface Induce AD10 T Cell IL2 Production, Independent of Endogenous or Null IEk, or IAk (A) Bio-IEk-MCC, bio-IEk-ER60, bio-IEk-HSP70, or bio-IEk-99A at the indicated concentrations were added to 96-well plates precoated with streptavidin and incubated for 18 h at 37 °C. AD10 CD4 + T cells were added to the wells and stimulated for 7 h prior to IL2 assay. (B) The level of IEk proteins on streptavidin plates coated as in (A) was determined by ELISA using anti-IEk 14-4-4s antibody followed by HRP-conjugated goat anti-mouse antibody. ABTS substrate color development was monitored at 405 nm. (C) Streptavidin plates coated with bio-IEk-MCC at the indicated concentrations were subsequently saturated with 10 μg/ml bio-IEk-ER60, bio-IEk-HSP70, bio-IEk-99A, or buffer for 1 h at room temperature before being used to stimulate AD10 T cells. (D) The 96-well ELISA plates were coated with bio-IEk-MCC at the indicated concentrations overnight at 4 °C. The wells were then saturated with 10 μg/ml BSA, IEk-ER60, IEk-HSP70, IEk-99A, or IAk-CA for 4 h at room temperature. Stimulation of AD10 and measurement of IL2 measurements were done as described in (A). (E and F) Same as (C) and (D), respectively, except that 5C.C7 cells were used.

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    22) Product Images from "Real-time single-molecule observation of rolling-circle DNA replication"

    Article Title: Real-time single-molecule observation of rolling-circle DNA replication

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp006

    ( a ) Schematic of single-molecule rolling-circle assay. ‘SA’, streptavidin. Upon leading-strand synthesis, DNA is displaced from the circle as the replisome ‘rolls’ around the template. The emerging ‘tail’ is converted to dsDNA via lagging-strand synthesis. As a result, the DNA that couples the M13 circle to the surface increases in length and is extended in the direction of flow. ( b ) Example field of view. Note both the length and number of products. Each flow cell has thousands of such fields, allowing for large numbers of products to be observed in a single experiment.
    Figure Legend Snippet: ( a ) Schematic of single-molecule rolling-circle assay. ‘SA’, streptavidin. Upon leading-strand synthesis, DNA is displaced from the circle as the replisome ‘rolls’ around the template. The emerging ‘tail’ is converted to dsDNA via lagging-strand synthesis. As a result, the DNA that couples the M13 circle to the surface increases in length and is extended in the direction of flow. ( b ) Example field of view. Note both the length and number of products. Each flow cell has thousands of such fields, allowing for large numbers of products to be observed in a single experiment.

    Techniques Used: Flow Cytometry

    23) Product Images from "Flagellar Hook Flexibility Is Essential for Bundle Formation in Swimming Escherichia coli Cells"

    Article Title: Flagellar Hook Flexibility Is Essential for Bundle Formation in Swimming Escherichia coli Cells

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00209-12

    Biophysical characterization of site A and site C AviTag mutants in response to streptavidin. (a) Hook stiffness of single tethered cells over time calculated using the equipartition theorem (see the text for details). Streptavidin (100 μM) was added at t = 5 min; stiffnesses are means ± standard errors of 28, 31, and 27 hooks from WT, site A, and site C strains, respectively. The inset shows the variance in body angle of a single cell from the site A data set versus time. (b) Analysis of free-swimming cells (see Videos S1, S2, and S3 in the supplemental material); histograms of the difference in angle between the cells' swimming trajectory and the angle of the cell body (illustrated by the inset). A cell swimming along its long axis gives an angle of 0°. (c) Selected video frames (3-ms exposure) of swimming cells dyed with Cy3 monofunctional succinimidyl ester showing the position of filaments. Scale bar, 3 μm (see Videos S4, S5, and S6).
    Figure Legend Snippet: Biophysical characterization of site A and site C AviTag mutants in response to streptavidin. (a) Hook stiffness of single tethered cells over time calculated using the equipartition theorem (see the text for details). Streptavidin (100 μM) was added at t = 5 min; stiffnesses are means ± standard errors of 28, 31, and 27 hooks from WT, site A, and site C strains, respectively. The inset shows the variance in body angle of a single cell from the site A data set versus time. (b) Analysis of free-swimming cells (see Videos S1, S2, and S3 in the supplemental material); histograms of the difference in angle between the cells' swimming trajectory and the angle of the cell body (illustrated by the inset). A cell swimming along its long axis gives an angle of 0°. (c) Selected video frames (3-ms exposure) of swimming cells dyed with Cy3 monofunctional succinimidyl ester showing the position of filaments. Scale bar, 3 μm (see Videos S4, S5, and S6).

    Techniques Used: Mass Spectrometry

    24) Product Images from "Padlock oligonucleotides as a tool for labeling superhelical DNA"

    Article Title: Padlock oligonucleotides as a tool for labeling superhelical DNA

    Journal: Nucleic Acids Research

    doi:

    Streptavidin binding to a biotinylated padlock oligonucleotide, as revealed by gel shift assay. A 5′-phosphorylated oligonucleotide with (lanes 3 and 4) or without (lanes 5–7) a biotinylated uridine was catenated to the pGA2 plasmid. Samples 2, 4, 6 and 7 were incubated in the presence of streptavidin before spermidine purification, as described in Materials and Methods. Samples were then incubated in the presence of 2 µM BQQ and digested with Dra III and Kpn I, analyzed on a 6% native polyacrylamide gel containing 10 mM MgCl 2 and revealed by SybrGreen I staining. In lane 7, a plasmid catenated to a non-biotinylated padlock was incubated in the presence of streptavidin and purified by spermidine compaction, then an equal amount of plasmid catenated to a biotinylated oligonucleotide was added to the sample before digestion. Bands I, II and III are described in the legend to Figure 2. IV corresponds to fragment II shifted by the presence of a biotinylated padlock oligonucleotide linked to streptavidin.
    Figure Legend Snippet: Streptavidin binding to a biotinylated padlock oligonucleotide, as revealed by gel shift assay. A 5′-phosphorylated oligonucleotide with (lanes 3 and 4) or without (lanes 5–7) a biotinylated uridine was catenated to the pGA2 plasmid. Samples 2, 4, 6 and 7 were incubated in the presence of streptavidin before spermidine purification, as described in Materials and Methods. Samples were then incubated in the presence of 2 µM BQQ and digested with Dra III and Kpn I, analyzed on a 6% native polyacrylamide gel containing 10 mM MgCl 2 and revealed by SybrGreen I staining. In lane 7, a plasmid catenated to a non-biotinylated padlock was incubated in the presence of streptavidin and purified by spermidine compaction, then an equal amount of plasmid catenated to a biotinylated oligonucleotide was added to the sample before digestion. Bands I, II and III are described in the legend to Figure 2. IV corresponds to fragment II shifted by the presence of a biotinylated padlock oligonucleotide linked to streptavidin.

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Plasmid Preparation, Incubation, Purification, Staining

    Images of the complex between the padlock oligonucleotide and the supercoiled pGA2 plasmid. ( a and b ) Annular dark field EM visualization of the biotinylated padlock oligonucleotide, revealed with gp32 single strand-binding protein (a) or streptavidin–ferritin conjugate (b). ( c and d ) The biotinylated padlock oligonucleotide was revealed with unmodified streptavidin using AFM. At high ionic strength (10 mM Tris, 5 mM MgCl 2 , 50 mM NaCl) (c) two supercoiled plectonemic forms complexed with streptavidin and a naked open circular form can be seen in the field. At low ionic strength (10 mM Tris, 5 mM MgCl 2 ) (d) the plasmid adopts a toroidal form and the streptavidin can be clearly seen. We cannot exclude the possibility that the plasmid shown here is not an open circular form, as it is not possible to distinguish the supercoiled and open circular forms under these conditions. The arrows indicate the position of the padlock oligonucleotide. The scale marker on EM views indicates 100 nm.
    Figure Legend Snippet: Images of the complex between the padlock oligonucleotide and the supercoiled pGA2 plasmid. ( a and b ) Annular dark field EM visualization of the biotinylated padlock oligonucleotide, revealed with gp32 single strand-binding protein (a) or streptavidin–ferritin conjugate (b). ( c and d ) The biotinylated padlock oligonucleotide was revealed with unmodified streptavidin using AFM. At high ionic strength (10 mM Tris, 5 mM MgCl 2 , 50 mM NaCl) (c) two supercoiled plectonemic forms complexed with streptavidin and a naked open circular form can be seen in the field. At low ionic strength (10 mM Tris, 5 mM MgCl 2 ) (d) the plasmid adopts a toroidal form and the streptavidin can be clearly seen. We cannot exclude the possibility that the plasmid shown here is not an open circular form, as it is not possible to distinguish the supercoiled and open circular forms under these conditions. The arrows indicate the position of the padlock oligonucleotide. The scale marker on EM views indicates 100 nm.

    Techniques Used: Plasmid Preparation, Binding Assay, Marker

    25) Product Images from "Preparation and binding study of a complex made of DNA-treated single-walled carbon nanotubes and antibody for specific delivery of a "molecular heater" platform"

    Article Title: Preparation and binding study of a complex made of DNA-treated single-walled carbon nanotubes and antibody for specific delivery of a "molecular heater" platform

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S34202

    Procedure for immobilization of streptavidin on the DNA-SWNT surface, and binding of biotinylated IgG to immobilized streptavidin to prepare the DNA-SWNT antibody complex. ( A ) Suspension of DNA-SWNT antibody complex. Abbreviations: Ab, antibody; SWNT, single-walled carbon nanotubes; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride.
    Figure Legend Snippet: Procedure for immobilization of streptavidin on the DNA-SWNT surface, and binding of biotinylated IgG to immobilized streptavidin to prepare the DNA-SWNT antibody complex. ( A ) Suspension of DNA-SWNT antibody complex. Abbreviations: Ab, antibody; SWNT, single-walled carbon nanotubes; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride.

    Techniques Used: Binding Assay

    Typical frequency changes for ( A ) streptavidin immobilized to DNA-SWNT according to the quartz crystal microbalance sensor, and ( B ) binding of biotinylated anti-human IgG to the streptavidin-immobilized DNA-SWNT according to the quartz crystal microbalance sensor. Abbreviation: SWNT, single-walled carbon nanotubes.
    Figure Legend Snippet: Typical frequency changes for ( A ) streptavidin immobilized to DNA-SWNT according to the quartz crystal microbalance sensor, and ( B ) binding of biotinylated anti-human IgG to the streptavidin-immobilized DNA-SWNT according to the quartz crystal microbalance sensor. Abbreviation: SWNT, single-walled carbon nanotubes.

    Techniques Used: Binding Assay

    26) Product Images from "Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP"

    Article Title: Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007956

    Pol V Mut binding to p/t DNA visualized at single-molecule resolution in real-time. (A) Sketch of smFRET experimental setup. An AF555 donor-labeled p/t DNA linked to streptavidin-biotin is attached to a glass slide surface. AF647 acceptor-labeled pol V Mut is then added, and DNA binding is observed as an increase in acceptor fluorophore emission that counter-correlates with a drop of a donor emission. (B) A representative smFRET trajectory showing multiple binding and unbinding events of ATPγS-activated pol V Mut E38K/ΔC17 (green = donor, red = acceptor, blue = FRET efficiency). ATPγS-activated pol V Mut was added at t = 30 s after the start of image acquisition. Data were collected for up to 3 min, prior to the onset of photobleaching. (C) Histogram representing smFRET efficiencies corresponding to the binding of ATPγS-activated pol V Mut E38K/ΔC17 to AF555-labeled p/t DNA. FRET efficiency is calculated as E = I A / (I D +I A ), where I A and I D represent acceptor and donor emission respectively. (D-F) Representative smFRET images are shown along with representative individual FRET trajectories of ATPγS-dependent binding of pol V Mut E38K/ΔC17 to p/t DNA. AF555-labeled p/t DNA is shown as green spots, and unbound AF647-labeled pol V Mut E38K/ΔC17 is shown as red spots. The pol V Mut E38K/ΔC17-p/t DNA binding events are shown as colocalized pol V Mut E38K/ΔC17 and p/t DNA signals (yellow/orange spots). Pol V Mut E38K/ΔC17 (D-E) or ATPγS activated pol V Mut E38K/ΔC17 (F) is added at t = 30 s after the start of image acquisition, followed by addition of ATPγS (t = 60 s, middle panel). Pol V Mut does not bind p/t DNA in the absence of ATPγS (D and S1 Movie ). The addition of ATPγS activates pol V Mut E38K/ΔC17, resulting in binding to p/t DNA (E and S2 Movie ) and pol V Mut E38K/ΔC17-p/t DNA binding events are indicated by the arrows. If pol V Mut is activated by ATPγS prior to addition to p/t DNA (F and S3 Movie ), multiple and rapid p/t DNA binding events occur, indicated by arrows. The images shown in (D-F) are smFRET data integrated over 1 min following pol V Mut addition (left panel) or first binding events (middle and right panels). Scale bar is 150 mm.
    Figure Legend Snippet: Pol V Mut binding to p/t DNA visualized at single-molecule resolution in real-time. (A) Sketch of smFRET experimental setup. An AF555 donor-labeled p/t DNA linked to streptavidin-biotin is attached to a glass slide surface. AF647 acceptor-labeled pol V Mut is then added, and DNA binding is observed as an increase in acceptor fluorophore emission that counter-correlates with a drop of a donor emission. (B) A representative smFRET trajectory showing multiple binding and unbinding events of ATPγS-activated pol V Mut E38K/ΔC17 (green = donor, red = acceptor, blue = FRET efficiency). ATPγS-activated pol V Mut was added at t = 30 s after the start of image acquisition. Data were collected for up to 3 min, prior to the onset of photobleaching. (C) Histogram representing smFRET efficiencies corresponding to the binding of ATPγS-activated pol V Mut E38K/ΔC17 to AF555-labeled p/t DNA. FRET efficiency is calculated as E = I A / (I D +I A ), where I A and I D represent acceptor and donor emission respectively. (D-F) Representative smFRET images are shown along with representative individual FRET trajectories of ATPγS-dependent binding of pol V Mut E38K/ΔC17 to p/t DNA. AF555-labeled p/t DNA is shown as green spots, and unbound AF647-labeled pol V Mut E38K/ΔC17 is shown as red spots. The pol V Mut E38K/ΔC17-p/t DNA binding events are shown as colocalized pol V Mut E38K/ΔC17 and p/t DNA signals (yellow/orange spots). Pol V Mut E38K/ΔC17 (D-E) or ATPγS activated pol V Mut E38K/ΔC17 (F) is added at t = 30 s after the start of image acquisition, followed by addition of ATPγS (t = 60 s, middle panel). Pol V Mut does not bind p/t DNA in the absence of ATPγS (D and S1 Movie ). The addition of ATPγS activates pol V Mut E38K/ΔC17, resulting in binding to p/t DNA (E and S2 Movie ) and pol V Mut E38K/ΔC17-p/t DNA binding events are indicated by the arrows. If pol V Mut is activated by ATPγS prior to addition to p/t DNA (F and S3 Movie ), multiple and rapid p/t DNA binding events occur, indicated by arrows. The images shown in (D-F) are smFRET data integrated over 1 min following pol V Mut addition (left panel) or first binding events (middle and right panels). Scale bar is 150 mm.

    Techniques Used: Binding Assay, Labeling

    27) Product Images from "Multiple cancer-specific antigens are targeted by a chimeric antigen receptor on a single cancer cell"

    Article Title: Multiple cancer-specific antigens are targeted by a chimeric antigen receptor on a single cancer cell

    Journal: JCI Insight

    doi: 10.1172/jci.insight.130416

    Recognition by 237CART cells is more permissive to amino acid residue substitutions and truncations of the Tn-glycopeptide epitope than that by 237Ab. Biotinylated peptides immobilized on a streptavidin-coated plate surface at the indicated coating concentrations were tested for binding by 237Ab measured by light absorption at 450 nm, or stimulation of 237CART cells measured by the level of IFN-γ secretion after 24 hours of coincubation. The Tn-glycosylated Thr 77 of murine PDPN is labeled in red, while the alanine scanning of the original murine PDPN sequence is labeled in green. Top panel: Alanine scanning of the 237Ab-binding epitope, 1 amino acid residue at a time. Middle panel: Alanine scanning of the 237Ab-binding epitope in murine PDPN with an increasing number of residues at a time. Bottom panel: The gradual truncation of the 237Ab-binding epitope from the C-terminus. Mean ± SEM, n = 3 from 3 independent experiments. The significance of the difference between each group at the highest peptide coating concentration in comparison to that of the non–Tn-glycosylated GTKPPLEE group was examined by 1-way ANOVA followed by Dunnett’s test. ns indicates P > 0.05; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
    Figure Legend Snippet: Recognition by 237CART cells is more permissive to amino acid residue substitutions and truncations of the Tn-glycopeptide epitope than that by 237Ab. Biotinylated peptides immobilized on a streptavidin-coated plate surface at the indicated coating concentrations were tested for binding by 237Ab measured by light absorption at 450 nm, or stimulation of 237CART cells measured by the level of IFN-γ secretion after 24 hours of coincubation. The Tn-glycosylated Thr 77 of murine PDPN is labeled in red, while the alanine scanning of the original murine PDPN sequence is labeled in green. Top panel: Alanine scanning of the 237Ab-binding epitope, 1 amino acid residue at a time. Middle panel: Alanine scanning of the 237Ab-binding epitope in murine PDPN with an increasing number of residues at a time. Bottom panel: The gradual truncation of the 237Ab-binding epitope from the C-terminus. Mean ± SEM, n = 3 from 3 independent experiments. The significance of the difference between each group at the highest peptide coating concentration in comparison to that of the non–Tn-glycosylated GTKPPLEE group was examined by 1-way ANOVA followed by Dunnett’s test. ns indicates P > 0.05; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

    Techniques Used: Binding Assay, Labeling, Sequencing, Concentration Assay

    28) Product Images from "Multiple cancer-specific antigens are targeted by a chimeric antigen receptor on a single cancer cell"

    Article Title: Multiple cancer-specific antigens are targeted by a chimeric antigen receptor on a single cancer cell

    Journal: JCI Insight

    doi: 10.1172/jci.insight.130416

    Recognition by 237CART cells is more permissive to amino acid residue substitutions and truncations of the Tn-glycopeptide epitope than that by 237Ab. Biotinylated peptides immobilized on a streptavidin-coated plate surface at the indicated coating concentrations were tested for binding by 237Ab measured by light absorption at 450 nm, or stimulation of 237CART cells measured by the level of IFN-γ secretion after 24 hours of coincubation. The Tn-glycosylated Thr 77 of murine PDPN is labeled in red, while the alanine scanning of the original murine PDPN sequence is labeled in green. Top panel: Alanine scanning of the 237Ab-binding epitope, 1 amino acid residue at a time. Middle panel: Alanine scanning of the 237Ab-binding epitope in murine PDPN with an increasing number of residues at a time. Bottom panel: The gradual truncation of the 237Ab-binding epitope from the C-terminus. Mean ± SEM, n = 3 from 3 independent experiments. The significance of the difference between each group at the highest peptide coating concentration in comparison to that of the non–Tn-glycosylated GTKPPLEE group was examined by 1-way ANOVA followed by Dunnett’s test. ns indicates P > 0.05; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
    Figure Legend Snippet: Recognition by 237CART cells is more permissive to amino acid residue substitutions and truncations of the Tn-glycopeptide epitope than that by 237Ab. Biotinylated peptides immobilized on a streptavidin-coated plate surface at the indicated coating concentrations were tested for binding by 237Ab measured by light absorption at 450 nm, or stimulation of 237CART cells measured by the level of IFN-γ secretion after 24 hours of coincubation. The Tn-glycosylated Thr 77 of murine PDPN is labeled in red, while the alanine scanning of the original murine PDPN sequence is labeled in green. Top panel: Alanine scanning of the 237Ab-binding epitope, 1 amino acid residue at a time. Middle panel: Alanine scanning of the 237Ab-binding epitope in murine PDPN with an increasing number of residues at a time. Bottom panel: The gradual truncation of the 237Ab-binding epitope from the C-terminus. Mean ± SEM, n = 3 from 3 independent experiments. The significance of the difference between each group at the highest peptide coating concentration in comparison to that of the non–Tn-glycosylated GTKPPLEE group was examined by 1-way ANOVA followed by Dunnett’s test. ns indicates P > 0.05; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

    Techniques Used: Binding Assay, Labeling, Sequencing, Concentration Assay

    29) Product Images from "Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection"

    Article Title: Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection

    Journal:

    doi: 10.1016/j.jsb.2008.07.008

    (A) Molecular representation of the hydrated streptavidin tetramer. (B) Projection map (32×32 pixels with a pixel size of 0.182 nm); (C) Projection map was Gaussian-filtered with a half power at 1/1.3 nm −1 ; (D) Data-derived projection
    Figure Legend Snippet: (A) Molecular representation of the hydrated streptavidin tetramer. (B) Projection map (32×32 pixels with a pixel size of 0.182 nm); (C) Projection map was Gaussian-filtered with a half power at 1/1.3 nm −1 ; (D) Data-derived projection

    Techniques Used: Derivative Assay

    Liposome tethering kinetics. EM images of negatively stained 2D streptavidin crystal (spanning holes in the perforated carbon film) with tethered liposomes (POPC liposomes doped with biotin-DPPE) at low (A), intermediate (B) and high (C–D) magnification.
    Figure Legend Snippet: Liposome tethering kinetics. EM images of negatively stained 2D streptavidin crystal (spanning holes in the perforated carbon film) with tethered liposomes (POPC liposomes doped with biotin-DPPE) at low (A), intermediate (B) and high (C–D) magnification.

    Techniques Used: Staining

    Protein concentration and crystal growth time effects on the crystal size. EM images show the negatively stained crystal grown from (A) 0.2 mg/ml (B) 0.1 mg/ml (C) 0.05 mg/ml of streptavidin solutions for 2 hours at room temperature. The black curves
    Figure Legend Snippet: Protein concentration and crystal growth time effects on the crystal size. EM images show the negatively stained crystal grown from (A) 0.2 mg/ml (B) 0.1 mg/ml (C) 0.05 mg/ml of streptavidin solutions for 2 hours at room temperature. The black curves

    Techniques Used: Protein Concentration, Staining

    30) Product Images from "Analysis of the DNA translocation and unwinding activities of T4 phage helicases"

    Article Title: Analysis of the DNA translocation and unwinding activities of T4 phage helicases

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2010.02.011

    Biotin–streptavidin block assay in the determination of UvsW translocation directionality. (A1 and B1) Schematic representation of the translocation of UvsW helicase on the biotin–streptavidin bearing DNA. Translocation of the enzyme away from the biotin–streptavidin block leads to rapid dissociation from the end of the DNA, whereas dissociation is prevented when translocation occurs in the direction of the block. ATPase activity of UvsW as a function of the 5′-biotin-labeled 30mer (A2) and 3′-biotin-labeled 30mer (B2) in the presence (closed circles) and absence (open circles) of streptavidin (1 μM).
    Figure Legend Snippet: Biotin–streptavidin block assay in the determination of UvsW translocation directionality. (A1 and B1) Schematic representation of the translocation of UvsW helicase on the biotin–streptavidin bearing DNA. Translocation of the enzyme away from the biotin–streptavidin block leads to rapid dissociation from the end of the DNA, whereas dissociation is prevented when translocation occurs in the direction of the block. ATPase activity of UvsW as a function of the 5′-biotin-labeled 30mer (A2) and 3′-biotin-labeled 30mer (B2) in the presence (closed circles) and absence (open circles) of streptavidin (1 μM).

    Techniques Used: Blocking Assay, Translocation Assay, Activity Assay, Labeling

    31) Product Images from "Screen Printed Carbon Electrode Based Electrochemical Immunosensor for the Detection of Dengue NS1 Antigen"

    Article Title: Screen Printed Carbon Electrode Based Electrochemical Immunosensor for the Detection of Dengue NS1 Antigen

    Journal: Diagnostics

    doi: 10.3390/diagnostics4040165

    Cyclic voltammetries of the unmodified and modified SPCEs: ( a ) unmodified SPCEs, ( b ) SPCEs/CMD, ( c ) SPCEs/CMD/streptavidin, ( d ) SPCEs/CMD/streptavidin/biotinylated anti-NS1capture antibody, ( e ) SPCEs/CMD/streptavidin/biotinylated anti-NS1 capture antibody/glycine, ( f ) SPCEs/CMD/streptavidin/biotinylated anti-NS1capture antibody/glycine/NS1 antigen.
    Figure Legend Snippet: Cyclic voltammetries of the unmodified and modified SPCEs: ( a ) unmodified SPCEs, ( b ) SPCEs/CMD, ( c ) SPCEs/CMD/streptavidin, ( d ) SPCEs/CMD/streptavidin/biotinylated anti-NS1capture antibody, ( e ) SPCEs/CMD/streptavidin/biotinylated anti-NS1 capture antibody/glycine, ( f ) SPCEs/CMD/streptavidin/biotinylated anti-NS1capture antibody/glycine/NS1 antigen.

    Techniques Used: Modification

    Immobilization of anti-NS1 capture antibody via passive adsorption and streptavidin/biotin system.
    Figure Legend Snippet: Immobilization of anti-NS1 capture antibody via passive adsorption and streptavidin/biotin system.

    Techniques Used: Adsorption

    32) Product Images from "Specific Protein Detection Using Designed DNA Carriers and Nanopores"

    Article Title: Specific Protein Detection Using Designed DNA Carriers and Nanopores

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja512521w

    Analysis of detection efficiency for different numbers of bound proteins. (a) A 400 μs window is created at the center of the translocation. The threshold Δ I (from the one double strand DNA level) is varied, and in (b) the percentage of translocations exceeding Δ I in the 400 μs window is calculated. The percentage is shown for designs 5B, 3B, 1B and 0B with 5, 3, 1, and 0 streptavidin attached, respectively. Error bars are the standard deviation from N independent nanopores with N = 3 (for 5B and 1B), N = 5 (for 3B) and N = 6 (for 0B) (raw values given in Tables S2–S5 ).
    Figure Legend Snippet: Analysis of detection efficiency for different numbers of bound proteins. (a) A 400 μs window is created at the center of the translocation. The threshold Δ I (from the one double strand DNA level) is varied, and in (b) the percentage of translocations exceeding Δ I in the 400 μs window is calculated. The percentage is shown for designs 5B, 3B, 1B and 0B with 5, 3, 1, and 0 streptavidin attached, respectively. Error bars are the standard deviation from N independent nanopores with N = 3 (for 5B and 1B), N = 5 (for 3B) and N = 6 (for 0B) (raw values given in Tables S2–S5 ).

    Techniques Used: Translocation Assay, Standard Deviation

    An assay for selective protein detection on a DNA carrier. (a) Events that begin and end with the one DNA double strand level are selected. If the current exceeds 50 pA from the baseline one DNA strand level within a central 400 μs window, the event is labeled positive for protein detection. Left shows an example of a positive translocation, right shows an example of a negative translocation. (b) Two DNA carrier designs used for experiment and controls. Three biotin (3B) design is as in Figure 3 with 3 biotin tags at the center. No modifications (0B) design has no biotin groups. Each design was incubated with one of two mixtures: mix1 contained the target streptavidin protein and mix2 contained BSA as a substitute control. A high percentage of threshold crossing events is only observed for the correct combination of binding site (biotin) and target protein (streptavidin). Error bars are the standard deviation from four independent nanopores (raw data is given in Tables S6–S9 ).
    Figure Legend Snippet: An assay for selective protein detection on a DNA carrier. (a) Events that begin and end with the one DNA double strand level are selected. If the current exceeds 50 pA from the baseline one DNA strand level within a central 400 μs window, the event is labeled positive for protein detection. Left shows an example of a positive translocation, right shows an example of a negative translocation. (b) Two DNA carrier designs used for experiment and controls. Three biotin (3B) design is as in Figure 3 with 3 biotin tags at the center. No modifications (0B) design has no biotin groups. Each design was incubated with one of two mixtures: mix1 contained the target streptavidin protein and mix2 contained BSA as a substitute control. A high percentage of threshold crossing events is only observed for the correct combination of binding site (biotin) and target protein (streptavidin). Error bars are the standard deviation from four independent nanopores (raw data is given in Tables S6–S9 ).

    Techniques Used: Labeling, Translocation Assay, Incubation, Binding Assay, Standard Deviation

    Adaptable binding site positions and chemistry. (a) DNA carrier design with three biotins separated at approximately one-quarter intervals along the DNA carrier. (b) Translocations after incubation of design (a) with streptavidin showing three spikes at approximately one-quarter, two quarters and three-quarters of the total translocation time. (c) Design of a DNA carrier with a digoxigenin tag at the central position and bound to an antidigoxigenin antibody. (d) Typical translocations after incubation with antidigoxigenin showing the presence of a current spike in the center. (e) Percentage of translocation exceeding Δ I from one DNA strand level (as in Figure 3 ). Error bars show the standard deviation from three independent nanopores with raw values given in Table S10 .
    Figure Legend Snippet: Adaptable binding site positions and chemistry. (a) DNA carrier design with three biotins separated at approximately one-quarter intervals along the DNA carrier. (b) Translocations after incubation of design (a) with streptavidin showing three spikes at approximately one-quarter, two quarters and three-quarters of the total translocation time. (c) Design of a DNA carrier with a digoxigenin tag at the central position and bound to an antidigoxigenin antibody. (d) Typical translocations after incubation with antidigoxigenin showing the presence of a current spike in the center. (e) Percentage of translocation exceeding Δ I from one DNA strand level (as in Figure 3 ). Error bars show the standard deviation from three independent nanopores with raw values given in Table S10 .

    Techniques Used: Binding Assay, Incubation, Translocation Assay, Standard Deviation

    Tailoring the number of binding sites on DNA carriers. (a), (b) and (c) show schematics of DNA carrier designs with 5, 3, and 1 biotin groups after incubation with streptavidin. For each design three typical translocation events are shown in (d), (e) and (f). Only events beginning and ending with one DNA double strand were selected. (g) The size and duration of the current spike in the center was measured relative to the double stranded DNA level. (h) Normalized histograms of the current change Δ I for the three designs. Each graph shows three lines which are independent nanopores. The minimum (threshold) Δ I was set to 40 pA. The total number of detected protein current spikes are 407 (5B), 288 (3B) and 166 (1B).
    Figure Legend Snippet: Tailoring the number of binding sites on DNA carriers. (a), (b) and (c) show schematics of DNA carrier designs with 5, 3, and 1 biotin groups after incubation with streptavidin. For each design three typical translocation events are shown in (d), (e) and (f). Only events beginning and ending with one DNA double strand were selected. (g) The size and duration of the current spike in the center was measured relative to the double stranded DNA level. (h) Normalized histograms of the current change Δ I for the three designs. Each graph shows three lines which are independent nanopores. The minimum (threshold) Δ I was set to 40 pA. The total number of detected protein current spikes are 407 (5B), 288 (3B) and 166 (1B).

    Techniques Used: Binding Assay, Incubation, Translocation Assay

    33) 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

    34) Product Images from "Whispering Gallery Modes in Standard Optical Fibres for Fibre Profiling Measurements and Sensing of Unlabelled Chemical Species"

    Article Title: Whispering Gallery Modes in Standard Optical Fibres for Fibre Profiling Measurements and Sensing of Unlabelled Chemical Species

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s100301765

    Frequency shift on binding of streptavidin to a biotinylated fibre. The spectrum of the biotinylated fibre is shown before and after the introduction of streptavidin to the sample cell as the dashed and solid lines, respectively.
    Figure Legend Snippet: Frequency shift on binding of streptavidin to a biotinylated fibre. The spectrum of the biotinylated fibre is shown before and after the introduction of streptavidin to the sample cell as the dashed and solid lines, respectively.

    Techniques Used: Binding Assay

    35) Product Images from "Small molecule detection with aptamer based lateral flow assays: Applying aptamer-C-reactive protein cross-recognition for ampicillin detection"

    Article Title: Small molecule detection with aptamer based lateral flow assays: Applying aptamer-C-reactive protein cross-recognition for ampicillin detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23963-6

    Schematic illustration of the designed competitive LFA for ampicillin detection. Aptamer-mFc-AuNP conjugates are incubated with biotinylated CRP in the presence or absence of ampicillin. If ampicillin is absent, the conjugates will bind towards biotinylated CRP and will be immobilized on the test line with streptavidin ( A ). If ampicillin is present, CRP is displaced from the aptamer, leading to a signal decrease on the test line ( B ). In both cases, the mFc on the conjugates leads to an immobilization on the control line via the α-mouse antibody, generating the control line.
    Figure Legend Snippet: Schematic illustration of the designed competitive LFA for ampicillin detection. Aptamer-mFc-AuNP conjugates are incubated with biotinylated CRP in the presence or absence of ampicillin. If ampicillin is absent, the conjugates will bind towards biotinylated CRP and will be immobilized on the test line with streptavidin ( A ). If ampicillin is present, CRP is displaced from the aptamer, leading to a signal decrease on the test line ( B ). In both cases, the mFc on the conjugates leads to an immobilization on the control line via the α-mouse antibody, generating the control line.

    Techniques Used: Incubation

    36) Product Images from "Neural Stem Cell Spreading on Lipid Based Artificial Cell Surfaces, Characterized by Combined X-ray and Neutron Reflectometry"

    Article Title: Neural Stem Cell Spreading on Lipid Based Artificial Cell Surfaces, Characterized by Combined X-ray and Neutron Reflectometry

    Journal: Materials

    doi: 10.3390/ma3114994

    Schematics. ( a ) Sample structure. The lipid bilayer containing 2% of biotinylated lipids (1) is covered by a layer of streptavidin (2) which is bound to the lipid membrane by biotin anchors. A biotinylated adhesion peptide (AK-cyclo[RGDfC]) is bound on top of the streptavidin layer (3); ( b ) Chemical structure of the cyclic RGD-containing binding residue; ( c ) Architecture of the adhesion peptide. (b) and (c) are adapted from [ 2 ].
    Figure Legend Snippet: Schematics. ( a ) Sample structure. The lipid bilayer containing 2% of biotinylated lipids (1) is covered by a layer of streptavidin (2) which is bound to the lipid membrane by biotin anchors. A biotinylated adhesion peptide (AK-cyclo[RGDfC]) is bound on top of the streptavidin layer (3); ( b ) Chemical structure of the cyclic RGD-containing binding residue; ( c ) Architecture of the adhesion peptide. (b) and (c) are adapted from [ 2 ].

    Techniques Used: Binding Assay

    Scattering length density and hydration profile. ( a ) Hydration profile of the layers including a schematic of the layer components. d hh indicates the head to head distance of the lipid bilayer; ( b ) Sld profiles of layers. Colored arrows indicate the respective y-axis; ( c ) Schematic of the synthetic peptide on top of the streptavidin interlayer. Red hooks depict the biotin residues, blue circles indicate the positions of a cyclo(RGDfC) residue. The bilayer-streptavidin interlayer is omitted. The table compares experimental findings and literature values.
    Figure Legend Snippet: Scattering length density and hydration profile. ( a ) Hydration profile of the layers including a schematic of the layer components. d hh indicates the head to head distance of the lipid bilayer; ( b ) Sld profiles of layers. Colored arrows indicate the respective y-axis; ( c ) Schematic of the synthetic peptide on top of the streptavidin interlayer. Red hooks depict the biotin residues, blue circles indicate the positions of a cyclo(RGDfC) residue. The bilayer-streptavidin interlayer is omitted. The table compares experimental findings and literature values.

    Techniques Used:

    Reflectometry data and best fits of the SLB/streptavidin /AK-cyclo[RGD] trilayer in liquid environment ( Figure 1 a). ( a ) The reflected intensity is plotted against the momentum transfer q z . Intensity scale is logarithmic and normalized to a total reflection signal of 1. The reflectometry data are represented by open symbols, curves represent best fits. The neutron measurements are shown in green [ D 2 O ] and red [ cm ], the X-ray measurement is shown in blue; ( b ) Expanded view of the neutron data and best fits. Colors and symbols are as in (a).
    Figure Legend Snippet: Reflectometry data and best fits of the SLB/streptavidin /AK-cyclo[RGD] trilayer in liquid environment ( Figure 1 a). ( a ) The reflected intensity is plotted against the momentum transfer q z . Intensity scale is logarithmic and normalized to a total reflection signal of 1. The reflectometry data are represented by open symbols, curves represent best fits. The neutron measurements are shown in green [ D 2 O ] and red [ cm ], the X-ray measurement is shown in blue; ( b ) Expanded view of the neutron data and best fits. Colors and symbols are as in (a).

    Techniques Used:

    37) Product Images from "Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter"

    Article Title: Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

    Journal: eLife

    doi: 10.7554/eLife.27283

    NiCl 2 mediated DNA deposition on Mica. DNA–end labeling via Streptavidin binding to the DNA biotin-tag. ( a, b ) Shows the length distributions for traced mtDNA and pUC18 DNA fragments in the presence of TFAM. The graphs show the contour lengths of each segment from individual DNA molecules. Each DNA molecule was traced starting with the attached Streptavidin label attached at the biotin-tag of the DNA fragment. The individual tracings have been sorted by the length of the long segment of the DNA starting with the Streptavidin labeled end. The center of the loop is indicated with a black bar. In ( a ) the orange box indicates the region containing the three mitochondrial promotors including the IPR region (~740 bp span). 39.1% of all counted loops on mtDNA molecules have been detected in this region (n = 44 of 113). ( b ) 18.2% of detected loops are present within this corresponding region of pUC18 DNA molecules (n = 6 of 33). DOI: http://dx.doi.org/10.7554/eLife.27283.019
    Figure Legend Snippet: NiCl 2 mediated DNA deposition on Mica. DNA–end labeling via Streptavidin binding to the DNA biotin-tag. ( a, b ) Shows the length distributions for traced mtDNA and pUC18 DNA fragments in the presence of TFAM. The graphs show the contour lengths of each segment from individual DNA molecules. Each DNA molecule was traced starting with the attached Streptavidin label attached at the biotin-tag of the DNA fragment. The individual tracings have been sorted by the length of the long segment of the DNA starting with the Streptavidin labeled end. The center of the loop is indicated with a black bar. In ( a ) the orange box indicates the region containing the three mitochondrial promotors including the IPR region (~740 bp span). 39.1% of all counted loops on mtDNA molecules have been detected in this region (n = 44 of 113). ( b ) 18.2% of detected loops are present within this corresponding region of pUC18 DNA molecules (n = 6 of 33). DOI: http://dx.doi.org/10.7554/eLife.27283.019

    Techniques Used: End Labeling, Binding Assay, Labeling

    NiCl 2 mediated DNA-protein complex deposition on Mica. ( a ) The AFM image shows an individual mtDNA molecule with a Streptavidin DNA-end label and a TFAM- mediated loop. The bound Streptavidin is clearly visible at the biotin-tagged end of the mtDNA (white arrow #1). The height profile shows a height of 3.5–4 nm for the TFAM-mediated loop at the mtDNA crossing (white arrow #2). The mtDNA itself shows a height of ~2 nm which is consistent with the height expected for low force AFM imaging in liquid. A schematic of the tracing of the individual segments of the DNA molecule is shown in ( b ). The long segment, starting with the Streptavidin label (white arrow), spans over ~927 bp, the loop segment for ~341 bp and the short segment for ~410 bp (total ~1678 bp). ( c ) Bare/naked mtDNA. The height profile displays a DNA height of 2–2.5 nm. The profile for the Streptavidin label shows a height of ~5 nm. ( d ) The bar graph shows the average height derived from DNA alone, DNA cross overs only, TFAM mediated DNA loops and the Streptavidin label from mtDNA molecules (n = 10). The bare DNA only displays a mean of 1.8 nm (SD 0.2 nm), DNA cross over only a mean of 2.2 nm (SD 0.24 nm), TFAM mediated loops show a mean of 3.8 nm (SD 0.26 nm), and the Streptavidin label a mean of 5.1 nm (SD 0.3 nm). Color bars range from 0 to 5.0 nm for both AFM images. DOI: http://dx.doi.org/10.7554/eLife.27283.018
    Figure Legend Snippet: NiCl 2 mediated DNA-protein complex deposition on Mica. ( a ) The AFM image shows an individual mtDNA molecule with a Streptavidin DNA-end label and a TFAM- mediated loop. The bound Streptavidin is clearly visible at the biotin-tagged end of the mtDNA (white arrow #1). The height profile shows a height of 3.5–4 nm for the TFAM-mediated loop at the mtDNA crossing (white arrow #2). The mtDNA itself shows a height of ~2 nm which is consistent with the height expected for low force AFM imaging in liquid. A schematic of the tracing of the individual segments of the DNA molecule is shown in ( b ). The long segment, starting with the Streptavidin label (white arrow), spans over ~927 bp, the loop segment for ~341 bp and the short segment for ~410 bp (total ~1678 bp). ( c ) Bare/naked mtDNA. The height profile displays a DNA height of 2–2.5 nm. The profile for the Streptavidin label shows a height of ~5 nm. ( d ) The bar graph shows the average height derived from DNA alone, DNA cross overs only, TFAM mediated DNA loops and the Streptavidin label from mtDNA molecules (n = 10). The bare DNA only displays a mean of 1.8 nm (SD 0.2 nm), DNA cross over only a mean of 2.2 nm (SD 0.24 nm), TFAM mediated loops show a mean of 3.8 nm (SD 0.26 nm), and the Streptavidin label a mean of 5.1 nm (SD 0.3 nm). Color bars range from 0 to 5.0 nm for both AFM images. DOI: http://dx.doi.org/10.7554/eLife.27283.018

    Techniques Used: Imaging, Derivative Assay

    38) Product Images from "Mutation Screening Based on the Mechanical Properties of DNA Molecules Tethered to a Solid Surface"

    Article Title: Mutation Screening Based on the Mechanical Properties of DNA Molecules Tethered to a Solid Surface

    Journal: The journal of physical chemistry. B

    doi: 10.1021/jp909501h

    Plot of raw ( blue curve ) and fitted ( black curve ) data for a typical frequency response and energy dissipation of the resonator at its fifth harmonic resonance as a function of time during the immobilization of the streptavidin layer and the ssDNA film.
    Figure Legend Snippet: Plot of raw ( blue curve ) and fitted ( black curve ) data for a typical frequency response and energy dissipation of the resonator at its fifth harmonic resonance as a function of time during the immobilization of the streptavidin layer and the ssDNA film.

    Techniques Used:

    Schematic process for immobilizing ssDNA strands with a complex 3-D structure on a quartz crystal. (a) A monolayer of biotin-thiol is immobilized onto the Au film by covalently linking the thiol group to Au. (b) A layer of streptavidin binds to the biotin
    Figure Legend Snippet: Schematic process for immobilizing ssDNA strands with a complex 3-D structure on a quartz crystal. (a) A monolayer of biotin-thiol is immobilized onto the Au film by covalently linking the thiol group to Au. (b) A layer of streptavidin binds to the biotin

    Techniques Used:

    39) Product Images from "Gene Related to Anergy in Lymphocytes (GRAIL) Expression in CD4+ T Cells Impairs Actin Cytoskeletal Organization during T Cell/Antigen-presenting Cell Interactions *"

    Article Title: Gene Related to Anergy in Lymphocytes (GRAIL) Expression in CD4+ T Cells Impairs Actin Cytoskeletal Organization during T Cell/Antigen-presenting Cell Interactions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.024497

    LFA-1 polarization, but not adhesion to ICAM, is impaired in GRAIL-expressing T cells. A , DO11.10 T cells expressing GFP or GRAIL-IRES-GFP were allowed to interact with OVA 323–33 9 -pulsed A20 cells for 15 min, and immunofluorescence was performed as described under “Experimental Procedures.” Confocal images were obtained, and single T/APC conjugates were examined for LFA-1 polarization to the T/APC interface. B , GFP- or GRAIL-expressing DO11.10 T cells were allowed to adhere to ICAM-1Fc-coated plates either resting or following activation as described under “Experimental Procedures.” Cells were vigorously washed, and fluorescence was quantified. Representative mean ± S.E. of three independent experiments is shown. CMAC , 7-amino-4-chloromethylcoumarin; SA , streptavidin; PMA , phorbol 12-myristate 13-acetate.
    Figure Legend Snippet: LFA-1 polarization, but not adhesion to ICAM, is impaired in GRAIL-expressing T cells. A , DO11.10 T cells expressing GFP or GRAIL-IRES-GFP were allowed to interact with OVA 323–33 9 -pulsed A20 cells for 15 min, and immunofluorescence was performed as described under “Experimental Procedures.” Confocal images were obtained, and single T/APC conjugates were examined for LFA-1 polarization to the T/APC interface. B , GFP- or GRAIL-expressing DO11.10 T cells were allowed to adhere to ICAM-1Fc-coated plates either resting or following activation as described under “Experimental Procedures.” Cells were vigorously washed, and fluorescence was quantified. Representative mean ± S.E. of three independent experiments is shown. CMAC , 7-amino-4-chloromethylcoumarin; SA , streptavidin; PMA , phorbol 12-myristate 13-acetate.

    Techniques Used: Expressing, Immunofluorescence, Activation Assay, Fluorescence

    Proximal TCR signaling is intact in GRAIL-expressing T cells. A , DO11.10 T cells expressing GFP alone or GRAIL-IRES-GFP were loaded with Indo-1-AM and incubated with biotinylated anti-mouse CD3. Baseline fluorescence was acquired for 30 s. Intracellular calcium mobilization was induced by cross-linking with streptavidin. Ionomycin was added to control for loading and viability ( inset ). B , DO11.10 T cells were incubated in serum-free media for 2 h at 37° C and left untreated or stained with biotinylated anti-CD3 followed by cross-linking with streptavidin. Cell lysates were subjected to electrophoresis, blots were probed with antibody to phosphorylated Vav ( pVav ), stripped, and reprobed with antibodies to total Vav ( B , top ). Band intensities were quantified and are displayed as normalized band volumes ( B , bottom ). Data are representative of four experiments.
    Figure Legend Snippet: Proximal TCR signaling is intact in GRAIL-expressing T cells. A , DO11.10 T cells expressing GFP alone or GRAIL-IRES-GFP were loaded with Indo-1-AM and incubated with biotinylated anti-mouse CD3. Baseline fluorescence was acquired for 30 s. Intracellular calcium mobilization was induced by cross-linking with streptavidin. Ionomycin was added to control for loading and viability ( inset ). B , DO11.10 T cells were incubated in serum-free media for 2 h at 37° C and left untreated or stained with biotinylated anti-CD3 followed by cross-linking with streptavidin. Cell lysates were subjected to electrophoresis, blots were probed with antibody to phosphorylated Vav ( pVav ), stripped, and reprobed with antibodies to total Vav ( B , top ). Band intensities were quantified and are displayed as normalized band volumes ( B , bottom ). Data are representative of four experiments.

    Techniques Used: Expressing, Incubation, Fluorescence, Staining, Electrophoresis

    GRAIL-expressing T cells have diminished JNK phosphorylation. DO11.10 T cells expressing GFP or GRAIL-IRES-GFP were incubated in serum-free media for 2 h at 37° C and left untreated or coated with biotinylated anti-CD3 followed by cross-linking with streptavidin for 5 min. Cell lysates were subjected to electrophoresis. Blots were then probed with antibodies to phosphorylated ERK ( A ) or phosphorylated JNK ( B ), stripped, and reprobed with antibodies to total ERK or JNK ( left ). Band intensities were quantified and are displayed as normalized band volumes ( right ). Data are representative of a minimum of three experiments.
    Figure Legend Snippet: GRAIL-expressing T cells have diminished JNK phosphorylation. DO11.10 T cells expressing GFP or GRAIL-IRES-GFP were incubated in serum-free media for 2 h at 37° C and left untreated or coated with biotinylated anti-CD3 followed by cross-linking with streptavidin for 5 min. Cell lysates were subjected to electrophoresis. Blots were then probed with antibodies to phosphorylated ERK ( A ) or phosphorylated JNK ( B ), stripped, and reprobed with antibodies to total ERK or JNK ( left ). Band intensities were quantified and are displayed as normalized band volumes ( right ). Data are representative of a minimum of three experiments.

    Techniques Used: Expressing, Incubation, Electrophoresis

    40) Product Images from "Nanodiamonds as multi-purpose labels for microscopy"

    Article Title: Nanodiamonds as multi-purpose labels for microscopy

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00797-2

    Multimodal analysis of FND70 immunolabeling of EpCAM-GFP HT29 cells. ( A ) Streptavidin-conjugated FND70 labels at the outside of a HT29 cell cluster. The context of different cells within the cluster is shown by GFP. Negative controls (left column) and positive controls (right column; QD655) are shown. No FND70 labeling of non-transfected negative controls is observed, context is shown by diffraction interference contrast (DIC) as EpCAM-GFP is absent. ( B ) Magnetic resonance spectra where taken at the bright spots identified as diamonds. The lower part of the figure shows the spectrum taken at the circled spot. 2,5% is the contrast between the resonance line and the background for 1 run. ( C ) CL, SE and the CL overlaid with BSE images of FND70 labeling an HT29 cell cluster at the cell surface. Note that in the right image single FND70 particles are resolved with CL. Abbreviations as in Fig. 3 . Bars: ( A , B ) 25 μm, ( C ) 1 μm.
    Figure Legend Snippet: Multimodal analysis of FND70 immunolabeling of EpCAM-GFP HT29 cells. ( A ) Streptavidin-conjugated FND70 labels at the outside of a HT29 cell cluster. The context of different cells within the cluster is shown by GFP. Negative controls (left column) and positive controls (right column; QD655) are shown. No FND70 labeling of non-transfected negative controls is observed, context is shown by diffraction interference contrast (DIC) as EpCAM-GFP is absent. ( B ) Magnetic resonance spectra where taken at the bright spots identified as diamonds. The lower part of the figure shows the spectrum taken at the circled spot. 2,5% is the contrast between the resonance line and the background for 1 run. ( C ) CL, SE and the CL overlaid with BSE images of FND70 labeling an HT29 cell cluster at the cell surface. Note that in the right image single FND70 particles are resolved with CL. Abbreviations as in Fig. 3 . Bars: ( A , B ) 25 μm, ( C ) 1 μm.

    Techniques Used: Immunolabeling, Labeling, Transfection

    FND applications in this study. ( a ) Representation of FND40 uptake by macrophages. The diamond particles are phagocytosed by the macrophages and are transported in intracellular vesicles. ( b ) Immunolabeling approach: (1) the extracellular domain of EpCAM, with an intracellular GFP domain, is targeted by a monoclonal antibody (MOC-31); (2) biotinylated rabbit anti mouse IgG is used as a linker for labeling with (3) streptavidin-conjugated FND70 particles. ( c ) Schematic overview of the sample area of a diamond magnetometer for intracellular sensing. The cells, which contain diamond nanoparticles, are in a glass bottom petri dish. A microwire in close proximity is used to excite in the microwave regime. Simultaneously, fluorescence is collected through a microscope objective and a subsequent confocal microscope. ( d ) Schematic overview of integrated light and scanning EM and cathodoluminescence. (0) Primary incident electrons generate (1) backscattered electrons (BSE) and (2) secondary electrons (SE) which can be imaged in a SEM with the respective detectors. Also photons can be generated upon electron beam excitation called (3) cathodoluminescence (CL). Via an optical lens, these photons can be detected with for example a CCD camera or photo multiplier tube (PMT). Furthermore, with an integrated light and electron microscope (4) regular fluorescence imaging can be performed with photon excitation.
    Figure Legend Snippet: FND applications in this study. ( a ) Representation of FND40 uptake by macrophages. The diamond particles are phagocytosed by the macrophages and are transported in intracellular vesicles. ( b ) Immunolabeling approach: (1) the extracellular domain of EpCAM, with an intracellular GFP domain, is targeted by a monoclonal antibody (MOC-31); (2) biotinylated rabbit anti mouse IgG is used as a linker for labeling with (3) streptavidin-conjugated FND70 particles. ( c ) Schematic overview of the sample area of a diamond magnetometer for intracellular sensing. The cells, which contain diamond nanoparticles, are in a glass bottom petri dish. A microwire in close proximity is used to excite in the microwave regime. Simultaneously, fluorescence is collected through a microscope objective and a subsequent confocal microscope. ( d ) Schematic overview of integrated light and scanning EM and cathodoluminescence. (0) Primary incident electrons generate (1) backscattered electrons (BSE) and (2) secondary electrons (SE) which can be imaged in a SEM with the respective detectors. Also photons can be generated upon electron beam excitation called (3) cathodoluminescence (CL). Via an optical lens, these photons can be detected with for example a CCD camera or photo multiplier tube (PMT). Furthermore, with an integrated light and electron microscope (4) regular fluorescence imaging can be performed with photon excitation.

    Techniques Used: Immunolabeling, Labeling, Fluorescence, Microscopy, Generated, Imaging

    41) Product Images from "Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity"

    Article Title: Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    SDS-PAGE of pET32a-stv-transformed cell lysates. Lane M, protein marker; Lane 1, cell lysates before induction; Lanes 2 and 3, cell lysates 2 and 4 h after induction, respectively. Recombinant streptavidin appears as a 36-kDa band in lanes 2 and 3.
    Figure Legend Snippet: SDS-PAGE of pET32a-stv-transformed cell lysates. Lane M, protein marker; Lane 1, cell lysates before induction; Lanes 2 and 3, cell lysates 2 and 4 h after induction, respectively. Recombinant streptavidin appears as a 36-kDa band in lanes 2 and 3.

    Techniques Used: SDS Page, Transformation Assay, Marker, Recombinant

    Amino acid sequence of streptavidin. The first 24 amino acids that comprise the signal peptide and inhibit biotin binding were removed. A: The full-length protein sequence. B: The protein sequence minus the signal peptide.
    Figure Legend Snippet: Amino acid sequence of streptavidin. The first 24 amino acids that comprise the signal peptide and inhibit biotin binding were removed. A: The full-length protein sequence. B: The protein sequence minus the signal peptide.

    Techniques Used: Sequencing, Binding Assay

    SDS-PAGE of Ni-NTA-purified recombinant streptavidin. Lane 1, protein marker; Lane 2, Ni-NTA-purified streptavidin.
    Figure Legend Snippet: SDS-PAGE of Ni-NTA-purified recombinant streptavidin. Lane 1, protein marker; Lane 2, Ni-NTA-purified streptavidin.

    Techniques Used: SDS Page, Purification, Recombinant, Marker

    Comparison of reading the optical density and Molar ratio of biotin to streptavidin in different condition of dialysis, according to table 1.S*: commercial strepatavidin (Sigma)
    Figure Legend Snippet: Comparison of reading the optical density and Molar ratio of biotin to streptavidin in different condition of dialysis, according to table 1.S*: commercial strepatavidin (Sigma)

    Techniques Used:

    42) Product Images from "Measurements of attractive forces between proteins and end-grafted poly(ethylene glycol) chains"

    Article Title: Measurements of attractive forces between proteins and end-grafted poly(ethylene glycol) chains

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

    doi:

    Force vs. distance profiles were measured during initial approach between streptavidin and DSPE-EO 45 monolayers at different polymer grafting densities. Experiments were performed at pH 7.0 and 25°C in 10 mM NaH 2 PO4 and 30 mM KNO 3 . The different EO 45 grafting densities were 480 (•), 960 (▪), and 2,280 (○) Å 2 per site, which corresponded to 9.0 (•), 4.5 (▪), and 1.5 (○) mol % DSPE-EO 45 in the DSPE matrix.
    Figure Legend Snippet: Force vs. distance profiles were measured during initial approach between streptavidin and DSPE-EO 45 monolayers at different polymer grafting densities. Experiments were performed at pH 7.0 and 25°C in 10 mM NaH 2 PO4 and 30 mM KNO 3 . The different EO 45 grafting densities were 480 (•), 960 (▪), and 2,280 (○) Å 2 per site, which corresponded to 9.0 (•), 4.5 (▪), and 1.5 (○) mol % DSPE-EO 45 in the DSPE matrix.

    Techniques Used:

    43) Product Images from "Dabigatran and Argatroban Diametrically Modulate Thrombin Exosite Function"

    Article Title: Dabigatran and Argatroban Diametrically Modulate Thrombin Exosite Function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157471

    Binding of thrombin to immobilized GpIbα269-286ppp. A . Increasing concentrations of thrombin up 4 μM were injected at a rate of 25 μl/min into flow cells of a streptavidin-coated CM5 biosensor chip containing adsorbed biotinlylated GpIbα269-286ppp. Response units (RU) are plotted versus time for the indicated thrombin concentrations (in nM) in triplicate (inset). RU values at equilibrium (Req) were determined and plotted versus the input thrombin concentration. The data were analyzed by nonlinear regression (line) to a rectangular hyperbola to determine K d . Symbols represent mean ± SD for from 3 runs. B . Thrombin (250 nM) was injected into flow cells containing immobilized biotinlylated GpIbα269-286ppp in the presence of 0–5000 nM dabigatran (circles), argatroban (triangles), or DAPA (squares). Req values were determined, converted to a fraction of that measured in the absence of inhibitor (Bound/Bound 0 ), and plotted versus the inhibitor concentration. Data points indicate mean ± SD for 3 determinations, and the lines represent nonlinear regression analyses.
    Figure Legend Snippet: Binding of thrombin to immobilized GpIbα269-286ppp. A . Increasing concentrations of thrombin up 4 μM were injected at a rate of 25 μl/min into flow cells of a streptavidin-coated CM5 biosensor chip containing adsorbed biotinlylated GpIbα269-286ppp. Response units (RU) are plotted versus time for the indicated thrombin concentrations (in nM) in triplicate (inset). RU values at equilibrium (Req) were determined and plotted versus the input thrombin concentration. The data were analyzed by nonlinear regression (line) to a rectangular hyperbola to determine K d . Symbols represent mean ± SD for from 3 runs. B . Thrombin (250 nM) was injected into flow cells containing immobilized biotinlylated GpIbα269-286ppp in the presence of 0–5000 nM dabigatran (circles), argatroban (triangles), or DAPA (squares). Req values were determined, converted to a fraction of that measured in the absence of inhibitor (Bound/Bound 0 ), and plotted versus the inhibitor concentration. Data points indicate mean ± SD for 3 determinations, and the lines represent nonlinear regression analyses.

    Techniques Used: Binding Assay, Injection, Flow Cytometry, Chromatin Immunoprecipitation, Concentration Assay

    44) Product Images from "A U87-EGFRvIII cell-specific aptamer mediates small interfering RNA delivery"

    Article Title: A U87-EGFRvIII cell-specific aptamer mediates small interfering RNA delivery

    Journal: Biomedical Reports

    doi: 10.3892/br.2014.276

    Comparison of the inhibitory rate on the proliferation of U87MG and U87-EGFRvIII cells following c-Met gene silencing. The inhibitory rate was calculated according to the OD values of the control and experimental groups obtained from the MTT assay. OD, optical density; BA, 5′-biotin-labeled aptamer 32; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + streptavidin; lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Data show the means ± standard deviation of three independent experiments. ** P
    Figure Legend Snippet: Comparison of the inhibitory rate on the proliferation of U87MG and U87-EGFRvIII cells following c-Met gene silencing. The inhibitory rate was calculated according to the OD values of the control and experimental groups obtained from the MTT assay. OD, optical density; BA, 5′-biotin-labeled aptamer 32; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + streptavidin; lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Data show the means ± standard deviation of three independent experiments. ** P

    Techniques Used: MTT Assay, Labeling, Small Interfering RNA, Standard Deviation

    Flow cytometry detection of apoptotic changes of U87-EGFRvIII cells following c-Met gene silencing. (A–D) Flow cytometry results of U87-EGFRvIII apoptosis in each group. Q2 and Q4 show late and early apoptotic rates, respectively. (E) Comparison of apoptotic rate of U87MG and U87-EGFRvIII cells in each group. BA, 5′-biotin-labeled aptamer 32; FITC, fluorescein isothiocyanate; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + streptavidin. lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Error bars show the means ± standard deviation (n=3). * P
    Figure Legend Snippet: Flow cytometry detection of apoptotic changes of U87-EGFRvIII cells following c-Met gene silencing. (A–D) Flow cytometry results of U87-EGFRvIII apoptosis in each group. Q2 and Q4 show late and early apoptotic rates, respectively. (E) Comparison of apoptotic rate of U87MG and U87-EGFRvIII cells in each group. BA, 5′-biotin-labeled aptamer 32; FITC, fluorescein isothiocyanate; siRNA, small interfering RNA; lipo + ncRNA + s = liposome + control siRNA + streptavidin. lipo + siRNA + s = liposome + c-Met siRNA + streptavidin; BA + ncRNA + s = BA + control siRNA + streptavidin; BA + siRNA + s = BA + c-Met siRNA + streptavidin. Error bars show the means ± standard deviation (n=3). * P

    Techniques Used: Flow Cytometry, Cytometry, Labeling, Small Interfering RNA, Standard Deviation

    BA-mediated delivery of c-Met siRNA into U87-EGFRvIII cells downregulated c-Met protein expression. (A) Determination of the amount of BA, c-Met siRNA (small interfering RNA) and streptavidin used for conjugation was performed by agarose gel electrophoresis. A32, biotin-aptamer 32 (BA); B-siRNA, biotin-c-Met siRNA; B-siRNA + s = biotin-c-Met siRNA + streptavidin. A32 + S + si = biotin-A32 + streptavidin + biotin-c-Met siRNA. (B) Western blotting and (C) quantitative analysis of the changes in c-Met protein expression following BA-delivered c-Met siRNA into U87-EGFRvIII cells. Transfected and lipo, liposome-mediated group; no transfection and BA group, BA-mediated group; NCsi = liposome or BA + control siRNA + streptavidin; Bsi + S = liposome or BA + c-Met siRNA + streptavidin; Bsi = liposome or BA + c-Met siRNA; BA = BA alone. Error bars show the means ± standard deviation (n=3). * P
    Figure Legend Snippet: BA-mediated delivery of c-Met siRNA into U87-EGFRvIII cells downregulated c-Met protein expression. (A) Determination of the amount of BA, c-Met siRNA (small interfering RNA) and streptavidin used for conjugation was performed by agarose gel electrophoresis. A32, biotin-aptamer 32 (BA); B-siRNA, biotin-c-Met siRNA; B-siRNA + s = biotin-c-Met siRNA + streptavidin. A32 + S + si = biotin-A32 + streptavidin + biotin-c-Met siRNA. (B) Western blotting and (C) quantitative analysis of the changes in c-Met protein expression following BA-delivered c-Met siRNA into U87-EGFRvIII cells. Transfected and lipo, liposome-mediated group; no transfection and BA group, BA-mediated group; NCsi = liposome or BA + control siRNA + streptavidin; Bsi + S = liposome or BA + c-Met siRNA + streptavidin; Bsi = liposome or BA + c-Met siRNA; BA = BA alone. Error bars show the means ± standard deviation (n=3). * P

    Techniques Used: Expressing, Small Interfering RNA, Conjugation Assay, Agarose Gel Electrophoresis, Western Blot, Transfection, Standard Deviation

    45) Product Images from "A comparison of 18F PET and 99mTc SPECT imaging in phantoms and in tumored mice"

    Article Title: A comparison of 18F PET and 99mTc SPECT imaging in phantoms and in tumored mice

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc1001467

    Showing the preparation scheme for 99m Tc and 18 F labeling streptavidin nanoparticles.
    Figure Legend Snippet: Showing the preparation scheme for 99m Tc and 18 F labeling streptavidin nanoparticles.

    Techniques Used: Labeling

    46) Product Images from "Discovery of Ligands for ?? Subunits from Phage-Displayed Peptide Libraries"

    Article Title: Discovery of Ligands for ?? Subunits from Phage-Displayed Peptide Libraries

    Journal: Methods in enzymology

    doi:

    Binding of phage displaying peptides to βγ subunits at various concentrations of phage particles. Fifty n M biotinylated βγ was bound to streptavidin-coated 96-well plate and various amounts of phage clones displaying two different peptide sequences were analyzed in the phage ELISA assay as is described in the text. Black bars are wild-type f88-4 phage controls; gray bars are two different clones displaying βγ binding peptides.
    Figure Legend Snippet: Binding of phage displaying peptides to βγ subunits at various concentrations of phage particles. Fifty n M biotinylated βγ was bound to streptavidin-coated 96-well plate and various amounts of phage clones displaying two different peptide sequences were analyzed in the phage ELISA assay as is described in the text. Black bars are wild-type f88-4 phage controls; gray bars are two different clones displaying βγ binding peptides.

    Techniques Used: Binding Assay, Clone Assay, Enzyme-linked Immunosorbent Assay

    47) Product Images from "Nanostructured Tip-Shaped Biosensors: Application of Six Sigma Approach for Enhanced Manufacturing"

    Article Title: Nanostructured Tip-Shaped Biosensors: Application of Six Sigma Approach for Enhanced Manufacturing

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s17010017

    Surface functionalization procedure of nanotip. The nanotip is modified by immersion into a solution containing 1 mg/mL streptavidin for 2 min. The streptavidin coated nanotip is then incubated with biotinylated LNA probe for 5 min.
    Figure Legend Snippet: Surface functionalization procedure of nanotip. The nanotip is modified by immersion into a solution containing 1 mg/mL streptavidin for 2 min. The streptavidin coated nanotip is then incubated with biotinylated LNA probe for 5 min.

    Techniques Used: Modification, Incubation

    ( a ) (Left) Diagram illustrating a nanotube tip covalently modified with a biotin ligand (dark-grey triangle) interacting with streptavidin protein receptors (light-grey blocks). (Right) Representative force–displacement curve recorded with a biotin-modified nanotube tip on the streptavidin surface in pH 7.0 PBS. The inset represents the scale bar. The binding force is indicated by F b . Reproduced with permission from reference [ 31 ]. Copyright 1998 Nature; ( b ) (Left) AFM topography image of a nanoelectrode. (right) Cyclic voltammograms obtained for different electrodes recorded in solutions of Fc(CH 2 OH) 2 and FcTMA. Reproduced with permission from reference [ 25 ]. Copyright 2006 American Chemical Society; ( c ) (Left) Schematic of nanoneedle biosensor three-dimensional and side view of horizontal nanoneedles (Not drawn to Scale). (Top right) Optical micrograph of bird’s eye view of aluminum-polysilicon hybrid nanoneedle biosensor. (Bottom right) SEM image of the tip of a nanoneedle biosensor. Reproduced with permission from reference [ 26 ]. Copyright 2013 American Institute of Physics; ( d ) A schematic of the pathway for generating the SERS platform for efficient Raman signal enhancement. Reproduced with permission from reference [ 14 ]. Copyright 2011 Elsevier B.V.
    Figure Legend Snippet: ( a ) (Left) Diagram illustrating a nanotube tip covalently modified with a biotin ligand (dark-grey triangle) interacting with streptavidin protein receptors (light-grey blocks). (Right) Representative force–displacement curve recorded with a biotin-modified nanotube tip on the streptavidin surface in pH 7.0 PBS. The inset represents the scale bar. The binding force is indicated by F b . Reproduced with permission from reference [ 31 ]. Copyright 1998 Nature; ( b ) (Left) AFM topography image of a nanoelectrode. (right) Cyclic voltammograms obtained for different electrodes recorded in solutions of Fc(CH 2 OH) 2 and FcTMA. Reproduced with permission from reference [ 25 ]. Copyright 2006 American Chemical Society; ( c ) (Left) Schematic of nanoneedle biosensor three-dimensional and side view of horizontal nanoneedles (Not drawn to Scale). (Top right) Optical micrograph of bird’s eye view of aluminum-polysilicon hybrid nanoneedle biosensor. (Bottom right) SEM image of the tip of a nanoneedle biosensor. Reproduced with permission from reference [ 26 ]. Copyright 2013 American Institute of Physics; ( d ) A schematic of the pathway for generating the SERS platform for efficient Raman signal enhancement. Reproduced with permission from reference [ 14 ]. Copyright 2011 Elsevier B.V.

    Techniques Used: Modification, Binding Assay

    48) Product Images from "Angular-Ratiometric Plasmon-Resonance Based Light Scattering for Bioaffinity Sensing"

    Article Title: Angular-Ratiometric Plasmon-Resonance Based Light Scattering for Bioaffinity Sensing

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja052739k

    Angular dependent scattering from 20 nm BSA-biotin colloids cross-linked by streptavidin (45 min incubation) from 532 nm (top) and 650 nm laser light (bottom).
    Figure Legend Snippet: Angular dependent scattering from 20 nm BSA-biotin colloids cross-linked by streptavidin (45 min incubation) from 532 nm (top) and 650 nm laser light (bottom).

    Techniques Used: Incubation

    Changes in absorption spectra of BSA-biotin colloids cross-linked by streptavidin (top) and the time dependent change in absorption at 600 nm for a 15 nM streptavidin addition (bottom).
    Figure Legend Snippet: Changes in absorption spectra of BSA-biotin colloids cross-linked by streptavidin (top) and the time dependent change in absorption at 600 nm for a 15 nM streptavidin addition (bottom).

    Techniques Used:

    I 140 / I 90 scattered intensity ratio from aggregated BSA-biotin colloids for both 532 and 650 nm laser light as a function of streptavidin concentration (45 min incubation time).
    Figure Legend Snippet: I 140 / I 90 scattered intensity ratio from aggregated BSA-biotin colloids for both 532 and 650 nm laser light as a function of streptavidin concentration (45 min incubation time).

    Techniques Used: Concentration Assay, Incubation

    49) Product Images from "In Vivo Delivery of Antisense MORF Oligomer by MORF/Carrier Streptavidin Nanoparticles"

    Article Title: In Vivo Delivery of Antisense MORF Oligomer by MORF/Carrier Streptavidin Nanoparticles

    Journal: Cancer Biotherapy & Radiopharmaceuticals

    doi: 10.1089/cbr.2009.0624

    Single-photon emission computed tomography/computed tomography images of a normal mouse at 30, 60, and 90 minutes postadministration of the 99m Tc-MORF/streptavidin/tat nanoparticle.
    Figure Legend Snippet: Single-photon emission computed tomography/computed tomography images of a normal mouse at 30, 60, and 90 minutes postadministration of the 99m Tc-MORF/streptavidin/tat nanoparticle.

    Techniques Used: Single Photon Emission Computed Tomography, Computed Tomography

    Radioactivity profiles by size-exclusion high-performance liquid chromatography. ( A ) 99m Tc-labeled antisense phosphorodiamidate morpholino (MORF). ( B ) 99m Tc-labeled antisense MORF/streptavidin nanoparticle before purification. ( C ) 99m Tc-labeled antisense
    Figure Legend Snippet: Radioactivity profiles by size-exclusion high-performance liquid chromatography. ( A ) 99m Tc-labeled antisense phosphorodiamidate morpholino (MORF). ( B ) 99m Tc-labeled antisense MORF/streptavidin nanoparticle before purification. ( C ) 99m Tc-labeled antisense

    Techniques Used: Radioactivity, High Performance Liquid Chromatography, Labeling, Purification

    50) Product Images from "Single-stranded DNA oligomers stimulate error-prone alternative repair of DNA double-strand breaks through hijacking Ku protein"

    Article Title: Single-stranded DNA oligomers stimulate error-prone alternative repair of DNA double-strand breaks through hijacking Ku protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv894

    Properties of ssDNA oligonucleotides to bind Ku and affect DNA repair proteins assembly at DNA ends. ( A ) Electrophoretic mobility shift assay of ds or ss biotinylated DNA probe incubated with various amount of purified human recombinant Ku protein. After acrylamide gel electrophoresis, transfer on membrane and UV-cross-linking, the position of free and retarded probe was revealed by chemiluminescence. ( B ) Electrophoretic mobility shift assay of ds biotinylated DNA probe incubated with purified human recombinant Ku protein in the presence of various amount of competitor ssO as indicated. ds probe and ssO amount in reactions were 0.01 pmol and from 0.3 to 10 pmol, respectively. Quantification of retarded probe in competition experiments as in (B) (mean ± SD, n = 3). ( C ) Electrophoretic mobility shift assay of radiolabeled ds DNA probe incubated with purified human recombinant Ku protein in the presence of various competitor ssO bearing a biotin moiety at the 3′-, 5′- or 3′- and 5′- end, blocked or not by streptavidin, as indicated. ds probe and ssO amount in reactions were 0.2 and 10 pmol, respectively. After acrylamide gel electrophoresis, the gel was dried and auto-radiographed. ( D ) Pull-down experiment on streptavidin magnetic beads bearing or not ds DNA probe, incubated with HCT116 cell extracts mixed or not with competitor ss- or ds-DNA, as indicated. ds probe and ssO amount in reactions were 0.8 pmol and 100 pmol, respectively. A fraction of the initial amount of protein used during the pull-down experiment was loaded as an input control. Proteins bound to the beads or remaining in the soluble fraction were denatured, separated on SDS-PAGE gel and electrotransferred on a membrane that was immunoblotted (IB) with the indicated antibodies.
    Figure Legend Snippet: Properties of ssDNA oligonucleotides to bind Ku and affect DNA repair proteins assembly at DNA ends. ( A ) Electrophoretic mobility shift assay of ds or ss biotinylated DNA probe incubated with various amount of purified human recombinant Ku protein. After acrylamide gel electrophoresis, transfer on membrane and UV-cross-linking, the position of free and retarded probe was revealed by chemiluminescence. ( B ) Electrophoretic mobility shift assay of ds biotinylated DNA probe incubated with purified human recombinant Ku protein in the presence of various amount of competitor ssO as indicated. ds probe and ssO amount in reactions were 0.01 pmol and from 0.3 to 10 pmol, respectively. Quantification of retarded probe in competition experiments as in (B) (mean ± SD, n = 3). ( C ) Electrophoretic mobility shift assay of radiolabeled ds DNA probe incubated with purified human recombinant Ku protein in the presence of various competitor ssO bearing a biotin moiety at the 3′-, 5′- or 3′- and 5′- end, blocked or not by streptavidin, as indicated. ds probe and ssO amount in reactions were 0.2 and 10 pmol, respectively. After acrylamide gel electrophoresis, the gel was dried and auto-radiographed. ( D ) Pull-down experiment on streptavidin magnetic beads bearing or not ds DNA probe, incubated with HCT116 cell extracts mixed or not with competitor ss- or ds-DNA, as indicated. ds probe and ssO amount in reactions were 0.8 pmol and 100 pmol, respectively. A fraction of the initial amount of protein used during the pull-down experiment was loaded as an input control. Proteins bound to the beads or remaining in the soluble fraction were denatured, separated on SDS-PAGE gel and electrotransferred on a membrane that was immunoblotted (IB) with the indicated antibodies.

    Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Purification, Recombinant, Acrylamide Gel Assay, Electrophoresis, Magnetic Beads, SDS Page

    Effect of ssDNA oligonucleotides on the recruitment and activation of DNA repair proteins at DSBs in cells. ( A ) Intracellular localization of LNA ssO 4 h after lipofection in HCT116 cells. a and c panels correspond to mock-transfected cells and b and d panels to transfected cells. Red images correspond to Cy5 fluorescence from LNA ssO and gray images to light transmission acquisition. Cells were visualized with an Olympus IX73 inverted fluorescence microscope equipped with a DP26 digital camera and a 20× objective lens. ( B ) Characterization of the damage response induced by cell transfection with oligonucleotides. The LNA ssO alone or annealed with the complementary strand (dsO) (150 pmol each per well) was transfected for 2 h into HCT116 cells in 6-well plates. After cell lysis, proteins were separated on SDS-PAGE gel and electro-transferred on a membrane that was blotted with the indicated antibodies. ( C ) Pull-down experiment. HCT116 cells were transfected with a biotinylated ds DNA ± LNA ssO as indicated. ds probe and ssO amount per well were 4 and 100 pmol, respectively. After cell lysis, the extracts were incubated with streptavidin magnetic beads. Proteins bound to the beads or remaining in the soluble fraction were denatured and separated on SDS-PAGE gel; then electro-transfer on membrane and immuno-blotting (IB) with the indicated antibodies were performed. ( D ) Effect of oligonucleotides on the cellular response to DSBs. The LNA ssO (150 pmol per well) was transfected for 2 h into HCT116 cells in 6-well plates. After washing, cells were incubated for 30 min in culture medium containing 80 pM Calicheamicin-γ1. After cell lysis, proteins were separated on SDS-PAGE gel and electro-transferred on a membrane that was blotted with the indicated antibodies.
    Figure Legend Snippet: Effect of ssDNA oligonucleotides on the recruitment and activation of DNA repair proteins at DSBs in cells. ( A ) Intracellular localization of LNA ssO 4 h after lipofection in HCT116 cells. a and c panels correspond to mock-transfected cells and b and d panels to transfected cells. Red images correspond to Cy5 fluorescence from LNA ssO and gray images to light transmission acquisition. Cells were visualized with an Olympus IX73 inverted fluorescence microscope equipped with a DP26 digital camera and a 20× objective lens. ( B ) Characterization of the damage response induced by cell transfection with oligonucleotides. The LNA ssO alone or annealed with the complementary strand (dsO) (150 pmol each per well) was transfected for 2 h into HCT116 cells in 6-well plates. After cell lysis, proteins were separated on SDS-PAGE gel and electro-transferred on a membrane that was blotted with the indicated antibodies. ( C ) Pull-down experiment. HCT116 cells were transfected with a biotinylated ds DNA ± LNA ssO as indicated. ds probe and ssO amount per well were 4 and 100 pmol, respectively. After cell lysis, the extracts were incubated with streptavidin magnetic beads. Proteins bound to the beads or remaining in the soluble fraction were denatured and separated on SDS-PAGE gel; then electro-transfer on membrane and immuno-blotting (IB) with the indicated antibodies were performed. ( D ) Effect of oligonucleotides on the cellular response to DSBs. The LNA ssO (150 pmol per well) was transfected for 2 h into HCT116 cells in 6-well plates. After washing, cells were incubated for 30 min in culture medium containing 80 pM Calicheamicin-γ1. After cell lysis, proteins were separated on SDS-PAGE gel and electro-transferred on a membrane that was blotted with the indicated antibodies.

    Techniques Used: Activation Assay, Transfection, Fluorescence, Transmission Assay, Microscopy, Lysis, SDS Page, Incubation, Magnetic Beads

    51) Product Images from "Antibodies to a strain-specific citrullinated Epstein-Barr virus peptide diagnoses rheumatoid arthritis"

    Article Title: Antibodies to a strain-specific citrullinated Epstein-Barr virus peptide diagnoses rheumatoid arthritis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22058-6

    Reactivity of rheumatoid arthritis sera and healthy donor sera to substituted and truncated linear EBNA-2 peptides analysed by streptavidin capture ELISA. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to healthy controls is used for comparison. ( a ) Reactivity of rheumatoid arthritis sera to EBNA-2 peptides (n = 10). ( b ) Reactivity of healthy donor sera to EBNA-2 peptides (n = 10).
    Figure Legend Snippet: Reactivity of rheumatoid arthritis sera and healthy donor sera to substituted and truncated linear EBNA-2 peptides analysed by streptavidin capture ELISA. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to healthy controls is used for comparison. ( a ) Reactivity of rheumatoid arthritis sera to EBNA-2 peptides (n = 10). ( b ) Reactivity of healthy donor sera to EBNA-2 peptides (n = 10).

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Reactivity of rheumatoid arthritis sera and healthy donor sera to linear and cyclic EBNA-2 peptides and control peptides analysed by streptavidin capture ELISA. ( a ) Reactivity of rheumatoid arthritis sera (n = 10) to cyclic and linear N/C-terminally biotinylated EBNA-2 peptides (amino acids 313–333 of Epstein-Barr virus strain AG876). “B” represents the location of the biotin labeling in relation the “EBNA” peptide. L = linear, C = cyclic. Linear peptide: GQGRGRWRG-Cit-GRSKGRGRMH-B, cyclic peptide: GQGRCGRWRG-Cit-GRSKGRGCRMH-B. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to healthy controls was used for comparison. ( b ) Reactivity of healthy donor sera (n = 10) to linear N/C-terminally biotinylated EBNA-2 peptides. ( c ) Reactivity of rheumatoid arthritis sera (n = 20) to linear and cyclic EBNA-2 peptide linked to a C-terminal biotin (amino acids 313–333 of Epstein-Barr virus strain AG876). Non-citrullinated peptides (Arg) were used at controls. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to non-citrullinated peptides is used for comparison. ( d ) Reactivity of healthy donor sera to linear and cyclic EBNA-2 peptides linked to a C-terminal biotin (n = 20).
    Figure Legend Snippet: Reactivity of rheumatoid arthritis sera and healthy donor sera to linear and cyclic EBNA-2 peptides and control peptides analysed by streptavidin capture ELISA. ( a ) Reactivity of rheumatoid arthritis sera (n = 10) to cyclic and linear N/C-terminally biotinylated EBNA-2 peptides (amino acids 313–333 of Epstein-Barr virus strain AG876). “B” represents the location of the biotin labeling in relation the “EBNA” peptide. L = linear, C = cyclic. Linear peptide: GQGRGRWRG-Cit-GRSKGRGRMH-B, cyclic peptide: GQGRCGRWRG-Cit-GRSKGRGCRMH-B. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to healthy controls was used for comparison. ( b ) Reactivity of healthy donor sera (n = 10) to linear N/C-terminally biotinylated EBNA-2 peptides. ( c ) Reactivity of rheumatoid arthritis sera (n = 20) to linear and cyclic EBNA-2 peptide linked to a C-terminal biotin (amino acids 313–333 of Epstein-Barr virus strain AG876). Non-citrullinated peptides (Arg) were used at controls. Statistical calculations were performed using the Student’s t-test, where antibody reactivity to non-citrullinated peptides is used for comparison. ( d ) Reactivity of healthy donor sera to linear and cyclic EBNA-2 peptides linked to a C-terminal biotin (n = 20).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Labeling

    Reactivity of rheumatoid arthritis and healthy donor sera to selected linear EBNA-2 peptides originating from three Epstein-Barr virus strains anslysed by streptavidin capture ELISA. *Specificity is calculated based on the reactivity of 10 healthy donor sera (HD) to the specific peptides. Statistical calculations are performed using the Student’s t-test, where antibody reactivity to healthy controls is used for comparison. a. Reactivity of rheumatoid arthritis sera (n = 15). b. Reactivity of healthy donor sera (n = 10).
    Figure Legend Snippet: Reactivity of rheumatoid arthritis and healthy donor sera to selected linear EBNA-2 peptides originating from three Epstein-Barr virus strains anslysed by streptavidin capture ELISA. *Specificity is calculated based on the reactivity of 10 healthy donor sera (HD) to the specific peptides. Statistical calculations are performed using the Student’s t-test, where antibody reactivity to healthy controls is used for comparison. a. Reactivity of rheumatoid arthritis sera (n = 15). b. Reactivity of healthy donor sera (n = 10).

    Techniques Used: Enzyme-linked Immunosorbent Assay

    52) Product Images from "Sizing the Protein Translocation Pathway of Colicin Ia Channels"

    Article Title: Sizing the Protein Translocation Pathway of Colicin Ia Channels

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200308852

    Structures of the stopper peptides. Each stopper is viewed along its longest axis, so that the smallest cross-sections are shown. The cross-sectional diameter, defined by the circumscribed circle (dashed line) is given for each stopper. (A) Peptide U, 12 Å. (B) α-Conotoxin ImI, 15 Å. (C) Apamin, 19 Å. (D) CTX, 26 Å. (E) Streptavidin tetramer bound to biotin, 61 Å. Details about the stopper structures and the estimation of their diameters are given in materials and methods . The pictures of animals in this and subsequent figures are provided to aid in matching each record with the corresponding stopper. In A–D, respectively, the animal represents the natural source of the stopper: unicorn–peptide U; cone snail–α-conotoxin; honeybee–apamin; scorpion–CTX.
    Figure Legend Snippet: Structures of the stopper peptides. Each stopper is viewed along its longest axis, so that the smallest cross-sections are shown. The cross-sectional diameter, defined by the circumscribed circle (dashed line) is given for each stopper. (A) Peptide U, 12 Å. (B) α-Conotoxin ImI, 15 Å. (C) Apamin, 19 Å. (D) CTX, 26 Å. (E) Streptavidin tetramer bound to biotin, 61 Å. Details about the stopper structures and the estimation of their diameters are given in materials and methods . The pictures of animals in this and subsequent figures are provided to aid in matching each record with the corresponding stopper. In A–D, respectively, the animal represents the natural source of the stopper: unicorn–peptide U; cone snail–α-conotoxin; honeybee–apamin; scorpion–CTX.

    Techniques Used:

    53) Product Images from "Regulation of Fyn Through Translocation of Activated Lck into Lipid Rafts"

    Article Title: Regulation of Fyn Through Translocation of Activated Lck into Lipid Rafts

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20022112

    Activation of lipid raft-associated Fyn is Lck dependent. TCRCβ and CD4 were coaggregated on CD4 + lymph node cells derived from wild-type (WT) or LGF + Lck −/− (Tg). (a) Total phosphotyrosyl content of each sample was assessed by immunoblotting with phophotyrosine-specific mAb (top panel). The filter was stripped and successively probed with anti-Lck (middle panel) followed by anti-Fyn (bottom panel). (b) Fyn immunoprecipitates were probed with pY418 Src and developed with Protein A-HRP (top panel). The filter was stripped and probed with anti-Fyn (middle panel). Quantification of pY417 Fyn normalized to total Fyn (bottom panel). (c) Distribution of GM1, Lck and Fyn in sucrose EDGC fractions derived from Tg cells subjected or not to streptavidin-mediated aggregation for 90 s. (d) Fyn immunoprecipitates derived from R and S fractions of WT and Tg cells were subjected to immune complex kinase assays. The phospho-enolase signals (pY Enolase) for Fyn (top panel) were normalized to total Fyn content (middle panel) and specific kinase activity was expressed as a ratio between pY Enolase and total Fyn (bottom panel). Bars representing R and S fractions from the same sample are grouped. Levels of pY417 Fyn as well as Fyn specific kinase activity in nonaggregated control samples were given a reference value “1.” Sample lanes in panels a and b are aligned over a common figure legend.
    Figure Legend Snippet: Activation of lipid raft-associated Fyn is Lck dependent. TCRCβ and CD4 were coaggregated on CD4 + lymph node cells derived from wild-type (WT) or LGF + Lck −/− (Tg). (a) Total phosphotyrosyl content of each sample was assessed by immunoblotting with phophotyrosine-specific mAb (top panel). The filter was stripped and successively probed with anti-Lck (middle panel) followed by anti-Fyn (bottom panel). (b) Fyn immunoprecipitates were probed with pY418 Src and developed with Protein A-HRP (top panel). The filter was stripped and probed with anti-Fyn (middle panel). Quantification of pY417 Fyn normalized to total Fyn (bottom panel). (c) Distribution of GM1, Lck and Fyn in sucrose EDGC fractions derived from Tg cells subjected or not to streptavidin-mediated aggregation for 90 s. (d) Fyn immunoprecipitates derived from R and S fractions of WT and Tg cells were subjected to immune complex kinase assays. The phospho-enolase signals (pY Enolase) for Fyn (top panel) were normalized to total Fyn content (middle panel) and specific kinase activity was expressed as a ratio between pY Enolase and total Fyn (bottom panel). Bars representing R and S fractions from the same sample are grouped. Levels of pY417 Fyn as well as Fyn specific kinase activity in nonaggregated control samples were given a reference value “1.” Sample lanes in panels a and b are aligned over a common figure legend.

    Techniques Used: Activation Assay, Derivative Assay, Immune Complex Kinase Assay, Activity Assay

    Coaggregation of TCR and CD4 induces the translocation of Lck into lipid rafts. (a) 10 7 primary CD4 + lymph node T cells were precoated with biotinylated anti-TCRCβ (H57 b ) and/or biotinylated anti-CD4 (GK1.5 b ) followed by the addition of Streptavidin for 10, 30, or 90 s, as indicated. Distribution of GM1, Lck, and Fyn was assessed in fractions 1–2 and 9–10, representing lipid raft (R) and soluble fractions (S), respectively. (b) Quantification of Lck subcellular distribution. Numbers represent the proportion of total Lck detected in R and S fractions. (c) Quantification of Lck enrichment in lipid rafts. The values represent the mean of three independent experiments performed as described for panel a with one SD indicated. (d) 10 7 primary CD4 + lymph node T cells were precoated with the indicated concentrations of H57 b and GK1.5 b and coaggregated or not by the addition of Streptavidin for 30 s. Distribution of Lck was assessed as described above. Quantification of the signals revealed was performed by densitometric analysis.
    Figure Legend Snippet: Coaggregation of TCR and CD4 induces the translocation of Lck into lipid rafts. (a) 10 7 primary CD4 + lymph node T cells were precoated with biotinylated anti-TCRCβ (H57 b ) and/or biotinylated anti-CD4 (GK1.5 b ) followed by the addition of Streptavidin for 10, 30, or 90 s, as indicated. Distribution of GM1, Lck, and Fyn was assessed in fractions 1–2 and 9–10, representing lipid raft (R) and soluble fractions (S), respectively. (b) Quantification of Lck subcellular distribution. Numbers represent the proportion of total Lck detected in R and S fractions. (c) Quantification of Lck enrichment in lipid rafts. The values represent the mean of three independent experiments performed as described for panel a with one SD indicated. (d) 10 7 primary CD4 + lymph node T cells were precoated with the indicated concentrations of H57 b and GK1.5 b and coaggregated or not by the addition of Streptavidin for 30 s. Distribution of Lck was assessed as described above. Quantification of the signals revealed was performed by densitometric analysis.

    Techniques Used: Translocation Assay

    Coaggregation of TCR and CD4 results in sequential activation of Lck then Fyn. (a) Total phosphotyrosyl content of each sample precoated with 1 μg/ml of biotinylated anti-TCRCβ (H57 b ( 1 )) and/or 0.3 μg/ml of biotinylated anti-CD4 (GK1.5 b (0.3)) and coaggregated with 50 μg/ml of streptavidin for 10, 30, and 90 s, was assessed by immuno-blotting with phosphotyrosine specific mAb. The filter was stripped and successively probed with anti-Lck followed by anti-Fyn. (b) Cell lysates probed with pY394 Lck. The filter was stripped and probed with anti-Lck. (c) Fyn immunoprecipitates were probed with pY418 Src. The filter was stripped and probed with anti-Fyn. Histograms in b and c show the quantification of pY394 Lck and pY417 Fyn normalized to total kinase signals. The nonaggregated control sample was given a reference value “1.” All sample lanes in a, b, and c are aligned over a common legend.
    Figure Legend Snippet: Coaggregation of TCR and CD4 results in sequential activation of Lck then Fyn. (a) Total phosphotyrosyl content of each sample precoated with 1 μg/ml of biotinylated anti-TCRCβ (H57 b ( 1 )) and/or 0.3 μg/ml of biotinylated anti-CD4 (GK1.5 b (0.3)) and coaggregated with 50 μg/ml of streptavidin for 10, 30, and 90 s, was assessed by immuno-blotting with phosphotyrosine specific mAb. The filter was stripped and successively probed with anti-Lck followed by anti-Fyn. (b) Cell lysates probed with pY394 Lck. The filter was stripped and probed with anti-Lck. (c) Fyn immunoprecipitates were probed with pY418 Src. The filter was stripped and probed with anti-Fyn. Histograms in b and c show the quantification of pY394 Lck and pY417 Fyn normalized to total kinase signals. The nonaggregated control sample was given a reference value “1.” All sample lanes in a, b, and c are aligned over a common legend.

    Techniques Used: Activation Assay

    54) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.039966

    Elongation of a human pMHC (gagSLY/HLA-A2) abrogates activation of G10 T cells expressing cognate TCR. A , schematic representation of single-chain constructs of native and elongated single-chain versions of HLA-A2/gag and corresponding coreceptor binding mutants ( yellow stars ). Constructs were stably expressed in TAP2-deficient CHO cells and sorted for comparable expression for use as surrogate antigen-presenting cells ( supplemental Fig. S3 A ). Also shown are soluble forms of native and elongated constructs comprising the extracellular portion followed by a C-terminal biotin acceptor site ( red dot ) and His 6 tag ( green line ). B , activation of primed G10 T cells was assessed by incubating 10 4 G10 cells/well with varying numbers of SCT-expressing APC (denoted as APC/G10 ratio) and IFNγ release measured by ELISA of culture supernatants after 8 h incubation. A representative plot of three independent experiments is shown. C , the human T cell clone SLY10 was used to assess responses to SCTs with mutated coreceptor-binding sites. Varying numbers of SCT-expressing CHO cells were incubated with 10 4 SLY10 cells/well, and MIP1b release was measured by ELISA of culture supernatants after 24 h of incubation. D , coreceptor binding to native and elongated SCTs was assessed by surface plasmon resonance (BIAcore). Monomeric biotinylated SCTs were immobilized by coupling to streptavidin conjugated CM5 flow cells (∼500 reference units). Binding curves were obtained by injecting CD8αα at a range of concentrations (0.9–154 μ m ) over flow cell surfaces immobilized with native or elongated SCTs. Immobilized biotinylated polyclonal anti-hamster IgG antibody was used as a control surface. The experiments were performed at 25 °C at a flow rate of 5 μl/min. Plateau response units were plotted against CD8αα concentration, and K D values were obtained by nonlinear curve fitting. The data are representative of two independent experiments. E , binding of native and elongated SCTs to G10 TCR was compared by surface plasmon resonance as described for D . The binding curves were obtained by injecting G10 TCR (0.1–7 μ m ) over immobilized SCTs, HLA-A2/NY-ESO, in addition to an irrelevant antibody-bound control surface.
    Figure Legend Snippet: Elongation of a human pMHC (gagSLY/HLA-A2) abrogates activation of G10 T cells expressing cognate TCR. A , schematic representation of single-chain constructs of native and elongated single-chain versions of HLA-A2/gag and corresponding coreceptor binding mutants ( yellow stars ). Constructs were stably expressed in TAP2-deficient CHO cells and sorted for comparable expression for use as surrogate antigen-presenting cells ( supplemental Fig. S3 A ). Also shown are soluble forms of native and elongated constructs comprising the extracellular portion followed by a C-terminal biotin acceptor site ( red dot ) and His 6 tag ( green line ). B , activation of primed G10 T cells was assessed by incubating 10 4 G10 cells/well with varying numbers of SCT-expressing APC (denoted as APC/G10 ratio) and IFNγ release measured by ELISA of culture supernatants after 8 h incubation. A representative plot of three independent experiments is shown. C , the human T cell clone SLY10 was used to assess responses to SCTs with mutated coreceptor-binding sites. Varying numbers of SCT-expressing CHO cells were incubated with 10 4 SLY10 cells/well, and MIP1b release was measured by ELISA of culture supernatants after 24 h of incubation. D , coreceptor binding to native and elongated SCTs was assessed by surface plasmon resonance (BIAcore). Monomeric biotinylated SCTs were immobilized by coupling to streptavidin conjugated CM5 flow cells (∼500 reference units). Binding curves were obtained by injecting CD8αα at a range of concentrations (0.9–154 μ m ) over flow cell surfaces immobilized with native or elongated SCTs. Immobilized biotinylated polyclonal anti-hamster IgG antibody was used as a control surface. The experiments were performed at 25 °C at a flow rate of 5 μl/min. Plateau response units were plotted against CD8αα concentration, and K D values were obtained by nonlinear curve fitting. The data are representative of two independent experiments. E , binding of native and elongated SCTs to G10 TCR was compared by surface plasmon resonance as described for D . The binding curves were obtained by injecting G10 TCR (0.1–7 μ m ) over immobilized SCTs, HLA-A2/NY-ESO, in addition to an irrelevant antibody-bound control surface.

    Techniques Used: Activation Assay, Expressing, Construct, Binding Assay, Stable Transfection, Enzyme-linked Immunosorbent Assay, Incubation, SPR Assay, Flow Cytometry, Concentration Assay

    TCR·CD3 triggering but not clustering is diminished by pMHC elongation. A , clean poly- l -lysine coated coverslips were incubated at 37 °C for 2 h with 50 μg/ml streptavidin/PBS. Biotinylated SCTs were immobilized on coverslips at comparable densities (Fig. S3 ). G10 cells were placed on SCT-coated coverslips and incubated for 1 min at 37 °C. The cells were fixed and permeabilized before serial staining CD3, Zap70, and CD45. The images were acquired using uniform settings between experiments. The cells adhered poorly on uncoated coverslips; therefore coverslips coated with biotinylated anti-HLA antibody was used as a control surface (without cognate TCR ligands). The scale bar represents 4 μm. B–D , the mean intensities for CD3, Zap70, and CD45 accumulation at the T cell interface was quantified. For a better appreciation of regions of TCR clustering pseudocolor scaled images of the CD3 distribution are shown (CD3 Scale) in A and E . The regions of CD3 clustering were arbitrarily defined by thresholding at two times the mean CD3 fluorescence intensity at the T cell interface (corresponding to the yellow regions in the pseudocolored scale images) and expressed as percentages of total CD3 interface fluorescence (Fig. S4 ). E , G10 cells were incubated with 10 μ m PP2 for 30 min prior to placing on to SCT-coated coverslips. Preparation and labeling were performed as described above. The scale bar represents 4 μm. The horizontal black bars represent the means. Statistical significance was determined by analysis of variance with correction for multiple comparisons. ns , p > 0.05; **, p
    Figure Legend Snippet: TCR·CD3 triggering but not clustering is diminished by pMHC elongation. A , clean poly- l -lysine coated coverslips were incubated at 37 °C for 2 h with 50 μg/ml streptavidin/PBS. Biotinylated SCTs were immobilized on coverslips at comparable densities (Fig. S3 ). G10 cells were placed on SCT-coated coverslips and incubated for 1 min at 37 °C. The cells were fixed and permeabilized before serial staining CD3, Zap70, and CD45. The images were acquired using uniform settings between experiments. The cells adhered poorly on uncoated coverslips; therefore coverslips coated with biotinylated anti-HLA antibody was used as a control surface (without cognate TCR ligands). The scale bar represents 4 μm. B–D , the mean intensities for CD3, Zap70, and CD45 accumulation at the T cell interface was quantified. For a better appreciation of regions of TCR clustering pseudocolor scaled images of the CD3 distribution are shown (CD3 Scale) in A and E . The regions of CD3 clustering were arbitrarily defined by thresholding at two times the mean CD3 fluorescence intensity at the T cell interface (corresponding to the yellow regions in the pseudocolored scale images) and expressed as percentages of total CD3 interface fluorescence (Fig. S4 ). E , G10 cells were incubated with 10 μ m PP2 for 30 min prior to placing on to SCT-coated coverslips. Preparation and labeling were performed as described above. The scale bar represents 4 μm. The horizontal black bars represent the means. Statistical significance was determined by analysis of variance with correction for multiple comparisons. ns , p > 0.05; **, p

    Techniques Used: Incubation, Staining, Fluorescence, Labeling

    55) Product Images from "Enhancing Nanoparticle-Based Visible Detection by Controlling the Extent of Aggregation"

    Article Title: Enhancing Nanoparticle-Based Visible Detection by Controlling the Extent of Aggregation

    Journal: Scientific Reports

    doi: 10.1038/srep00456

    (a) Scheme of the two-step assay using an SL designed in “conjugate” fashion for detecting a target with multiple binding sites. (b) Effect of switching SLs off on decreasing the extent of aggregation of NPs, when nLK determines the aggregation. (c) Effect of switching SLs off on increasing the extent of aggregation of NPs, when the number of unoccupied binding sites determines the aggregation. (d) Color of the test system with different amounts of bBSA at 2 h after performing the assay for detecting streptavidin using a known concentration of stAuNPs (absorption: 0.43 @ 531 nm for 1/10 diluted sample) (e) Test system scaled-down (one-half of d) with one-half concentration of stAuNPs (absorption: 0.21 @ 531 nm for 1/10 diluted sample), which enhances the detection sensitivity down to 2×10 -14 M.
    Figure Legend Snippet: (a) Scheme of the two-step assay using an SL designed in “conjugate” fashion for detecting a target with multiple binding sites. (b) Effect of switching SLs off on decreasing the extent of aggregation of NPs, when nLK determines the aggregation. (c) Effect of switching SLs off on increasing the extent of aggregation of NPs, when the number of unoccupied binding sites determines the aggregation. (d) Color of the test system with different amounts of bBSA at 2 h after performing the assay for detecting streptavidin using a known concentration of stAuNPs (absorption: 0.43 @ 531 nm for 1/10 diluted sample) (e) Test system scaled-down (one-half of d) with one-half concentration of stAuNPs (absorption: 0.21 @ 531 nm for 1/10 diluted sample), which enhances the detection sensitivity down to 2×10 -14 M.

    Techniques Used: Binding Assay, Concentration Assay

    56) Product Images from "Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity"

    Article Title: Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    SDS-PAGE of pET32a-stv-transformed cell lysates. Lane M, protein marker; Lane 1, cell lysates before induction; Lanes 2 and 3, cell lysates 2 and 4 h after induction, respectively. Recombinant streptavidin appears as a 36-kDa band in lanes 2 and 3.
    Figure Legend Snippet: SDS-PAGE of pET32a-stv-transformed cell lysates. Lane M, protein marker; Lane 1, cell lysates before induction; Lanes 2 and 3, cell lysates 2 and 4 h after induction, respectively. Recombinant streptavidin appears as a 36-kDa band in lanes 2 and 3.

    Techniques Used: SDS Page, Transformation Assay, Marker, Recombinant

    Amino acid sequence of streptavidin. The first 24 amino acids that comprise the signal peptide and inhibit biotin binding were removed. A: The full-length protein sequence. B: The protein sequence minus the signal peptide.
    Figure Legend Snippet: Amino acid sequence of streptavidin. The first 24 amino acids that comprise the signal peptide and inhibit biotin binding were removed. A: The full-length protein sequence. B: The protein sequence minus the signal peptide.

    Techniques Used: Sequencing, Binding Assay

    SDS-PAGE of Ni-NTA-purified recombinant streptavidin. Lane 1, protein marker; Lane 2, Ni-NTA-purified streptavidin.
    Figure Legend Snippet: SDS-PAGE of Ni-NTA-purified recombinant streptavidin. Lane 1, protein marker; Lane 2, Ni-NTA-purified streptavidin.

    Techniques Used: SDS Page, Purification, Recombinant, Marker

    Comparison of reading the optical density and Molar ratio of biotin to streptavidin in different condition of dialysis, according to table 1.S*: commercial strepatavidin (Sigma)
    Figure Legend Snippet: Comparison of reading the optical density and Molar ratio of biotin to streptavidin in different condition of dialysis, according to table 1.S*: commercial strepatavidin (Sigma)

    Techniques Used:

    57) Product Images from "Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity"

    Article Title: Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    SDS-PAGE of pET32a-stv-transformed cell lysates. Lane M, protein marker; Lane 1, cell lysates before induction; Lanes 2 and 3, cell lysates 2 and 4 h after induction, respectively. Recombinant streptavidin appears as a 36-kDa band in lanes 2 and 3.
    Figure Legend Snippet: SDS-PAGE of pET32a-stv-transformed cell lysates. Lane M, protein marker; Lane 1, cell lysates before induction; Lanes 2 and 3, cell lysates 2 and 4 h after induction, respectively. Recombinant streptavidin appears as a 36-kDa band in lanes 2 and 3.

    Techniques Used: SDS Page, Transformation Assay, Marker, Recombinant

    Amino acid sequence of streptavidin. The first 24 amino acids that comprise the signal peptide and inhibit biotin binding were removed. A: The full-length protein sequence. B: The protein sequence minus the signal peptide.
    Figure Legend Snippet: Amino acid sequence of streptavidin. The first 24 amino acids that comprise the signal peptide and inhibit biotin binding were removed. A: The full-length protein sequence. B: The protein sequence minus the signal peptide.

    Techniques Used: Sequencing, Binding Assay

    SDS-PAGE of Ni-NTA-purified recombinant streptavidin. Lane 1, protein marker; Lane 2, Ni-NTA-purified streptavidin.
    Figure Legend Snippet: SDS-PAGE of Ni-NTA-purified recombinant streptavidin. Lane 1, protein marker; Lane 2, Ni-NTA-purified streptavidin.

    Techniques Used: SDS Page, Purification, Recombinant, Marker

    Comparison of reading the optical density and Molar ratio of biotin to streptavidin in different condition of dialysis, according to table 1.S*: commercial strepatavidin (Sigma)
    Figure Legend Snippet: Comparison of reading the optical density and Molar ratio of biotin to streptavidin in different condition of dialysis, according to table 1.S*: commercial strepatavidin (Sigma)

    Techniques Used:

    58) Product Images from "Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity"

    Article Title: Production of Recombinant Streptavidin and Optimization of Refolding Conditions for Recovery of Biological Activity

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    SDS-PAGE of pET32a-stv-transformed cell lysates. Lane M, protein marker; Lane 1, cell lysates before induction; Lanes 2 and 3, cell lysates 2 and 4 h after induction, respectively. Recombinant streptavidin appears as a 36-kDa band in lanes 2 and 3.
    Figure Legend Snippet: SDS-PAGE of pET32a-stv-transformed cell lysates. Lane M, protein marker; Lane 1, cell lysates before induction; Lanes 2 and 3, cell lysates 2 and 4 h after induction, respectively. Recombinant streptavidin appears as a 36-kDa band in lanes 2 and 3.

    Techniques Used: SDS Page, Transformation Assay, Marker, Recombinant

    Amino acid sequence of streptavidin. The first 24 amino acids that comprise the signal peptide and inhibit biotin binding were removed. A: The full-length protein sequence. B: The protein sequence minus the signal peptide.
    Figure Legend Snippet: Amino acid sequence of streptavidin. The first 24 amino acids that comprise the signal peptide and inhibit biotin binding were removed. A: The full-length protein sequence. B: The protein sequence minus the signal peptide.

    Techniques Used: Sequencing, Binding Assay

    SDS-PAGE of Ni-NTA-purified recombinant streptavidin. Lane 1, protein marker; Lane 2, Ni-NTA-purified streptavidin.
    Figure Legend Snippet: SDS-PAGE of Ni-NTA-purified recombinant streptavidin. Lane 1, protein marker; Lane 2, Ni-NTA-purified streptavidin.

    Techniques Used: SDS Page, Purification, Recombinant, Marker

    Comparison of reading the optical density and Molar ratio of biotin to streptavidin in different condition of dialysis, according to table 1.S*: commercial strepatavidin (Sigma)
    Figure Legend Snippet: Comparison of reading the optical density and Molar ratio of biotin to streptavidin in different condition of dialysis, according to table 1.S*: commercial strepatavidin (Sigma)

    Techniques Used:

    59) Product Images from "Physical Characteristics of a Citrullinated Pro-Filaggrin Epitope Recognized by Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Sera"

    Article Title: Physical Characteristics of a Citrullinated Pro-Filaggrin Epitope Recognized by Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Sera

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0168542

    Reactivity of RA sera (n = 20) and healthy donor sera (n = 20) to linear and cyclic citrullinated peptides pro-filaggrin peptides analyzed by streptavidin capture ELISA. The peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template, whereas the non-citrullinated peptide was used as control. B-LCPa and LCPa-B represent peptides with a N- and C-terminal biotin labelling, respectively. A. Reactivity of RA sera to pro-filaggrin peptides. B. Reactivity of healthy donor sera to pro-filaggrin peptides.
    Figure Legend Snippet: Reactivity of RA sera (n = 20) and healthy donor sera (n = 20) to linear and cyclic citrullinated peptides pro-filaggrin peptides analyzed by streptavidin capture ELISA. The peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template, whereas the non-citrullinated peptide was used as control. B-LCPa and LCPa-B represent peptides with a N- and C-terminal biotin labelling, respectively. A. Reactivity of RA sera to pro-filaggrin peptides. B. Reactivity of healthy donor sera to pro-filaggrin peptides.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Reactivity of RA sera (n = 20) to lysine-substituted linear citrullinated pro-filaggrin peptides analyzed by streptavidin capture ELISA. The citrullinated peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template. LCPa was used as positive control.
    Figure Legend Snippet: Reactivity of RA sera (n = 20) to lysine-substituted linear citrullinated pro-filaggrin peptides analyzed by streptavidin capture ELISA. The citrullinated peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template. LCPa was used as positive control.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Positive Control

    Reactivity of RA sera (n = 20) to D-Ala substituted peptides analyzed by streptavidin capture ELISA. The linear peptide SHQEST-Cit-GRSRGRS was used as template, LCPb was used as control. D-Ala substitution was introduced at position 8.
    Figure Legend Snippet: Reactivity of RA sera (n = 20) to D-Ala substituted peptides analyzed by streptavidin capture ELISA. The linear peptide SHQEST-Cit-GRSRGRS was used as template, LCPb was used as control. D-Ala substitution was introduced at position 8.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Reactivity of RA sera (n = 20) to glycine-substituted linear citrullinated pro-filaggrin peptides analyzed by streptavidin capture ELISA. The citrullinated peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template. Non-citrullinated LCPg was used as negative controls.
    Figure Legend Snippet: Reactivity of RA sera (n = 20) to glycine-substituted linear citrullinated pro-filaggrin peptides analyzed by streptavidin capture ELISA. The citrullinated peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template. Non-citrullinated LCPg was used as negative controls.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Reactivity of RA sera (n = 20) and healthy donor sera (n = 20) to linear and cyclic citrullinated pro-filaggrin peptides analyzed by streptavidin capture ELISA. The peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template, whereas the non-citrullinated peptide was used as control. A. Reactivity of RA sera to N-terminal biotinylated linear peptides. B. Reactivity of healthy donor sera to N-terminal biotinylated linear pro-filaggrin peptides. C. Reactivity of RA sera to C-terminal biotinylated linear peptides. D. Reactivity of healthy donor sera to C-terminal biotinylated linear pro-filaggrin peptides. E. Reactivity of RA sera to cyclic peptides. F. Reactivity of healthy donor sera to cyclic pro-filaggrin peptides.
    Figure Legend Snippet: Reactivity of RA sera (n = 20) and healthy donor sera (n = 20) to linear and cyclic citrullinated pro-filaggrin peptides analyzed by streptavidin capture ELISA. The peptide HQCHQEST-Cit-GRSRGRCGRSGS was used as template, whereas the non-citrullinated peptide was used as control. A. Reactivity of RA sera to N-terminal biotinylated linear peptides. B. Reactivity of healthy donor sera to N-terminal biotinylated linear pro-filaggrin peptides. C. Reactivity of RA sera to C-terminal biotinylated linear peptides. D. Reactivity of healthy donor sera to C-terminal biotinylated linear pro-filaggrin peptides. E. Reactivity of RA sera to cyclic peptides. F. Reactivity of healthy donor sera to cyclic pro-filaggrin peptides.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    60) Product Images from "MINIMIZING ELECTROSTATIC CHARGING OF AN APERTURE USED TO PRODUCE IN-FOCUS PHASE CONTRAST IN THE TEM"

    Article Title: MINIMIZING ELECTROSTATIC CHARGING OF AN APERTURE USED TO PRODUCE IN-FOCUS PHASE CONTRAST IN THE TEM

    Journal: Ultramicroscopy

    doi: 10.1016/j.ultramic.2013.05.023

    Three successive images recorded at the same area of a streptavidin monolayer crystal, using an electron exposure of approximately 1500 electrons/nm 2 for each image. This series illustrates the fact that in-focus images of weak phase objects have high contrast when taken with the tulip aperture. While the second and third image in the series must suffer successively greater degrees of radiation damage, the effects of this damage are not expected to be apparent at the low resolution of features that can be seen by eye. The unit-cell dimensions of the streptavidin crystal are estimated to be a = 8.05 nm, b = 8.16 nm. (A) Image taken close to focus, with a well-centered tulip aperture. (B) Image taken without changing the focus value, but with the tulip aperture retracted. (C) Image again taken with the tulip aperture retracted, but after under-focusing the image by ~3.7 μm.
    Figure Legend Snippet: Three successive images recorded at the same area of a streptavidin monolayer crystal, using an electron exposure of approximately 1500 electrons/nm 2 for each image. This series illustrates the fact that in-focus images of weak phase objects have high contrast when taken with the tulip aperture. While the second and third image in the series must suffer successively greater degrees of radiation damage, the effects of this damage are not expected to be apparent at the low resolution of features that can be seen by eye. The unit-cell dimensions of the streptavidin crystal are estimated to be a = 8.05 nm, b = 8.16 nm. (A) Image taken close to focus, with a well-centered tulip aperture. (B) Image taken without changing the focus value, but with the tulip aperture retracted. (C) Image again taken with the tulip aperture retracted, but after under-focusing the image by ~3.7 μm.

    Techniques Used:

    Illustration of the effectiveness of image restoration. An image was selected that contains a streptavidin monolayer crystal on the left and a disordered monolayer of streptavidin molecules clustered at the top right. A nearly empty region of the lipid monolayer is present at the center and lower right of this image. Individual streptavidin tetramers (M r = 55 k), which are present within this sparsely populated area, can be seen in both the raw and the restored versions of the image. The unit-cell dimensions of the streptavidin crystal are estimated to be a = 8.05 nm, b = 8.16 nm. (A) The “raw” image, showing the “optical shadowing” effect that is present before restoration. (B) Restored image with arrows identify three examples of streptavidin tetramers. This image was produced by applying a systematic correction of 90 degrees to the phases for all spatial frequencies within the single-sideband region of the Fourier transform of the raw image. A quadratic phase-correction, rising to a value of 30 degrees at a spatial frequency of 1/(1.3 nm), was also applied as part of the restoration procedure.
    Figure Legend Snippet: Illustration of the effectiveness of image restoration. An image was selected that contains a streptavidin monolayer crystal on the left and a disordered monolayer of streptavidin molecules clustered at the top right. A nearly empty region of the lipid monolayer is present at the center and lower right of this image. Individual streptavidin tetramers (M r = 55 k), which are present within this sparsely populated area, can be seen in both the raw and the restored versions of the image. The unit-cell dimensions of the streptavidin crystal are estimated to be a = 8.05 nm, b = 8.16 nm. (A) The “raw” image, showing the “optical shadowing” effect that is present before restoration. (B) Restored image with arrows identify three examples of streptavidin tetramers. This image was produced by applying a systematic correction of 90 degrees to the phases for all spatial frequencies within the single-sideband region of the Fourier transform of the raw image. A quadratic phase-correction, rising to a value of 30 degrees at a spatial frequency of 1/(1.3 nm), was also applied as part of the restoration procedure.

    Techniques Used: Produced

    61) Product Images from "Detection and isolation of auto-reactive human antibodies from primary B cells"

    Article Title: Detection and isolation of auto-reactive human antibodies from primary B cells

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2013.06.018

    Characterization of ACPA. (A) CCP reactivity of donor plasma as determined by ELISA. Briefly, microtiter plates are coated with streptavidin, incubated first with biotinylated-CCP and then with plasma at different dilutions. Detection is enabled by incubation
    Figure Legend Snippet: Characterization of ACPA. (A) CCP reactivity of donor plasma as determined by ELISA. Briefly, microtiter plates are coated with streptavidin, incubated first with biotinylated-CCP and then with plasma at different dilutions. Detection is enabled by incubation

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation

    62) Product Images from "Synthesis of Water-Dispersed Ferrecene/Phenylboronic Acid-Modified Bifunctional Gold Nanoparticles and the Application in Biosensing"

    Article Title: Synthesis of Water-Dispersed Ferrecene/Phenylboronic Acid-Modified Bifunctional Gold Nanoparticles and the Application in Biosensing

    Journal: Materials

    doi: 10.3390/ma7085554

    ( A ) The fits with the Randles equivalent circuit; ( B ) electrochemical impedance spectroscopy (EIS) of bare (black curve), biotin-covered (red curve), biotin/MCH/BSA-covered (blue curve), and biotin/MCH/BSA/avidin-covered (olive curve) gold electrode; ( C ) CVs of the biotin/MCH/BSA/avidin-covered electrode before (black curve) and after (red curve) incubation with FBA solution; ( D ) CVs of the biotin/MCH/BSA-covered electrode with (black curve) and without (red curve) the capture of avidin, followed by the attachment of Fc–MBA–AuNPs. The blue curve in panel D corresponds to that with streptavidin in place of avidin. The concentration of avidin was 157 pM, and the arrow indicates the scan direction.
    Figure Legend Snippet: ( A ) The fits with the Randles equivalent circuit; ( B ) electrochemical impedance spectroscopy (EIS) of bare (black curve), biotin-covered (red curve), biotin/MCH/BSA-covered (blue curve), and biotin/MCH/BSA/avidin-covered (olive curve) gold electrode; ( C ) CVs of the biotin/MCH/BSA/avidin-covered electrode before (black curve) and after (red curve) incubation with FBA solution; ( D ) CVs of the biotin/MCH/BSA-covered electrode with (black curve) and without (red curve) the capture of avidin, followed by the attachment of Fc–MBA–AuNPs. The blue curve in panel D corresponds to that with streptavidin in place of avidin. The concentration of avidin was 157 pM, and the arrow indicates the scan direction.

    Techniques Used: Impedance Spectroscopy, Avidin-Biotin Assay, Incubation, Concentration Assay

    63) Product Images from "Single-molecule detection of DNA hybridization"

    Article Title: Single-molecule detection of DNA hybridization

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

    doi: 10.1073/pnas.1337215100

    ( a ) Effect of surface charges; the displacement of a bead when charges are added to its surface. The streptavidin-functionalized bead is held by several ssDNA tethers; when biotinylated DNA oligomers (18 mer) are added to the solution (10
    Figure Legend Snippet: ( a ) Effect of surface charges; the displacement of a bead when charges are added to its surface. The streptavidin-functionalized bead is held by several ssDNA tethers; when biotinylated DNA oligomers (18 mer) are added to the solution (10

    Techniques Used:

    64) Product Images from "A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin"

    Article Title: A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin

    Journal: Scientific Reports

    doi: 10.1038/srep21369

    Streptavidin binding test of liposomal aptamer ST-21 ( A ) and ST-21M ( B ). The Liposome only is negative control without aptamer-tagged. Both liposomal aptamers showed strong binding to streptavidin, indicating the successful fabrication of liposomal aptamers.
    Figure Legend Snippet: Streptavidin binding test of liposomal aptamer ST-21 ( A ) and ST-21M ( B ). The Liposome only is negative control without aptamer-tagged. Both liposomal aptamers showed strong binding to streptavidin, indicating the successful fabrication of liposomal aptamers.

    Techniques Used: Binding Assay, Negative Control

    The liposomal aptamer with the ability of binding to streptavidin. The cholesterol incorporated into lipid bilayer through hydrophobic interactions. The liposomes encapsulated hundreds of thousands of fluorescent dyes for providing strong signal to aptamer.
    Figure Legend Snippet: The liposomal aptamer with the ability of binding to streptavidin. The cholesterol incorporated into lipid bilayer through hydrophobic interactions. The liposomes encapsulated hundreds of thousands of fluorescent dyes for providing strong signal to aptamer.

    Techniques Used: Binding Assay

    Schematic diagram of our liposomal aptamer replacement assay for the detection of biotin. The liposomal aptamers were pre-bound to streptavidin coated on a 96-well plate. When biotin was presented in the sample, the liposomal aptamers will be replaced by biotin. Fluorescent signals were measured by a microtiter plate reader.
    Figure Legend Snippet: Schematic diagram of our liposomal aptamer replacement assay for the detection of biotin. The liposomal aptamers were pre-bound to streptavidin coated on a 96-well plate. When biotin was presented in the sample, the liposomal aptamers will be replaced by biotin. Fluorescent signals were measured by a microtiter plate reader.

    Techniques Used:

    The streptavidin-binding aptamers, ST-21 and ST-21M, with the modification of TEG and cholesterol. TEG spacer provides an easier approach for streptavidin-binding. Cholesterol was used to incorporate into lipid bilayer of liposomes. The asterisk indicates mutation site in the sequence of ST-21M.
    Figure Legend Snippet: The streptavidin-binding aptamers, ST-21 and ST-21M, with the modification of TEG and cholesterol. TEG spacer provides an easier approach for streptavidin-binding. Cholesterol was used to incorporate into lipid bilayer of liposomes. The asterisk indicates mutation site in the sequence of ST-21M.

    Techniques Used: Binding Assay, Modification, Mutagenesis, Sequencing

    65) Product Images from "Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation [S]"

    Article Title: Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M064089

    Lysosome exocytosis and acidification in extracellular compartments formed at sites of contact with apoptotic adipocytes. J774 cells (A–C) or huMDMs (D–F) were incubated overnight with biotin-fluorescein-dextran to deliver the dextran to lysosomes. Cells were then incubated with streptavidin-labeled UV-induced apoptotic 3T3 L1 adipocytes (A–C) or UV-induced apoptotic primary murine adipocytes (D–F) for 90 min followed by a 30 sec treatment with 200 μM biotin-Alexa546 to mark extracellular streptavidin-labeled structures. Cells were then fixed and permeabilized to remove unbound biotin-fluorescein-dextran from the lysosomes. Representative images of biotin-fluorescein-dextran (A, D), biotin-Alexa546 (B, E), and merged images superimposed on phase contrast images (C, F). Colocalization of the fluorescein and Alexa546 signals demonstrates that lysosomal contents are delivered to extracellular compartments (arrows). G–I: Macrophages were incubated with apoptotic adipocytes labeled with a pH-sensitive fluorophore and a pH-insensitive fluorophore. Ratiometric images showing acidified compartments as J774 cells interact with a TNF-α-induced apoptotic primary murine adipocyte (G), a huMDM interacts with a TNF-α-induced apoptotic primary murine adipocyte (H), and J774 cells interact with an UV-induced apoptotic 3T3 L1 adipocyte (I). Regions of low pH are contiguous with the adipocyte body, thus supporting that they are extracellular [insets, arrows (I)]. The lower inset is an enlarged image of the macrophage-adipocyte interface showing variations in pH. The upper inset shows the CypHer fluorescence of the same region as the lower inset and is included for clarity. J–N: TNF-α-induced apoptotic primary murine adipocytes were labeled with Alexa546 and incubated with J774 cells for 90 min in the absence (J, K) or presence (L, M) of bafilomycin A1, a V-ATPase inhibitor. Following the 90 min incubation, macrophages were labeled with Alexa488-CtB on ice, and the amount of Alexa546 signal within the macrophage was quantified. N: Bafilomycin A1 treatment abolished macrophage adipocyte uptake. Data are from three independent experiments. Error bars represent the SEM. *** P ≤ 0.001 Wilcoxon rank sum test. Size bars 20 μm. AD, adipocyte; M, macrophage.
    Figure Legend Snippet: Lysosome exocytosis and acidification in extracellular compartments formed at sites of contact with apoptotic adipocytes. J774 cells (A–C) or huMDMs (D–F) were incubated overnight with biotin-fluorescein-dextran to deliver the dextran to lysosomes. Cells were then incubated with streptavidin-labeled UV-induced apoptotic 3T3 L1 adipocytes (A–C) or UV-induced apoptotic primary murine adipocytes (D–F) for 90 min followed by a 30 sec treatment with 200 μM biotin-Alexa546 to mark extracellular streptavidin-labeled structures. Cells were then fixed and permeabilized to remove unbound biotin-fluorescein-dextran from the lysosomes. Representative images of biotin-fluorescein-dextran (A, D), biotin-Alexa546 (B, E), and merged images superimposed on phase contrast images (C, F). Colocalization of the fluorescein and Alexa546 signals demonstrates that lysosomal contents are delivered to extracellular compartments (arrows). G–I: Macrophages were incubated with apoptotic adipocytes labeled with a pH-sensitive fluorophore and a pH-insensitive fluorophore. Ratiometric images showing acidified compartments as J774 cells interact with a TNF-α-induced apoptotic primary murine adipocyte (G), a huMDM interacts with a TNF-α-induced apoptotic primary murine adipocyte (H), and J774 cells interact with an UV-induced apoptotic 3T3 L1 adipocyte (I). Regions of low pH are contiguous with the adipocyte body, thus supporting that they are extracellular [insets, arrows (I)]. The lower inset is an enlarged image of the macrophage-adipocyte interface showing variations in pH. The upper inset shows the CypHer fluorescence of the same region as the lower inset and is included for clarity. J–N: TNF-α-induced apoptotic primary murine adipocytes were labeled with Alexa546 and incubated with J774 cells for 90 min in the absence (J, K) or presence (L, M) of bafilomycin A1, a V-ATPase inhibitor. Following the 90 min incubation, macrophages were labeled with Alexa488-CtB on ice, and the amount of Alexa546 signal within the macrophage was quantified. N: Bafilomycin A1 treatment abolished macrophage adipocyte uptake. Data are from three independent experiments. Error bars represent the SEM. *** P ≤ 0.001 Wilcoxon rank sum test. Size bars 20 μm. AD, adipocyte; M, macrophage.

    Techniques Used: Incubation, Labeling, Size-exclusion Chromatography, Fluorescence, CtB Assay

    66) Product Images from "Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells"

    Article Title: Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells

    Journal: Nature Communications

    doi: 10.1038/ncomms11371

    Hypoxia down-regulates global membrane protein endocytosis. ( a ) HeLa cells were pre-treated at normoxia or hypoxia (1% O 2 ) for 20 h, followed by cell-surface biotinylation, staining with streptavidin-AF-488 and visualization by confocal microscopy. Scale bar, 20 μm. ( b ) FACS quantification of biotinylated cell-surface proteome in HeLa cells shows inhibition by hypoxic treatment for the indicated time periods. ( c ) Immunoblotting for biotinylated cell-surface proteins from a similar experiment as described in ( b ) shows down-regulation by hypoxia. ( d ) Confocal microscopy imaging of endocytosed, biotinylated membrane proteins following 30 min of internalization and cell-surface biotinylation depletion in HeLa cells shows inhibition by hypoxic treatment for the indicated time periods. Scale bar, 10 μm. ( e ) FACS quantification of the endocytosed membrane proteome from a similar experiment as described in ( d ) shows down-regulation by hypoxia. ( f ) Immunoblotting for endocytosed proteins from a similar experiment as described in ( d ) shows inhibition by hypoxia. ( g–l ) FACS quantification of biotinylated cell-surface proteome ( g – i ) and endocytosed membrane proteome ( j – l ) following 30 min of internalization performed with the indicated cell types (A549, lung adenocarcinoma; MDA-MB-231, breast adenocarcinoma; U87-MG, glioblastoma) treated at normoxia or hypoxia for 20 h. Data are presented as % relative to normoxic cells (in b and g–i ) or as % of total cell-surface biotinylation at normoxia and hypoxia, respectively, at t =0 (in e , and j–l )±s.d. from three independent experiments. * P
    Figure Legend Snippet: Hypoxia down-regulates global membrane protein endocytosis. ( a ) HeLa cells were pre-treated at normoxia or hypoxia (1% O 2 ) for 20 h, followed by cell-surface biotinylation, staining with streptavidin-AF-488 and visualization by confocal microscopy. Scale bar, 20 μm. ( b ) FACS quantification of biotinylated cell-surface proteome in HeLa cells shows inhibition by hypoxic treatment for the indicated time periods. ( c ) Immunoblotting for biotinylated cell-surface proteins from a similar experiment as described in ( b ) shows down-regulation by hypoxia. ( d ) Confocal microscopy imaging of endocytosed, biotinylated membrane proteins following 30 min of internalization and cell-surface biotinylation depletion in HeLa cells shows inhibition by hypoxic treatment for the indicated time periods. Scale bar, 10 μm. ( e ) FACS quantification of the endocytosed membrane proteome from a similar experiment as described in ( d ) shows down-regulation by hypoxia. ( f ) Immunoblotting for endocytosed proteins from a similar experiment as described in ( d ) shows inhibition by hypoxia. ( g–l ) FACS quantification of biotinylated cell-surface proteome ( g – i ) and endocytosed membrane proteome ( j – l ) following 30 min of internalization performed with the indicated cell types (A549, lung adenocarcinoma; MDA-MB-231, breast adenocarcinoma; U87-MG, glioblastoma) treated at normoxia or hypoxia for 20 h. Data are presented as % relative to normoxic cells (in b and g–i ) or as % of total cell-surface biotinylation at normoxia and hypoxia, respectively, at t =0 (in e , and j–l )±s.d. from three independent experiments. * P

    Techniques Used: Staining, Confocal Microscopy, FACS, Inhibition, Imaging, Multiple Displacement Amplification

    Encoding of hypoxia-induced internalizing surface proteins. ( a ) HeLa cells were pre-treated at normoxia or hypoxia for 20 h. Total surface proteins and internalized proteins, following 2 h of endocytosis, were isolated by streptavidin affinity chromatography and identified by LC–MS/MS analysis. Candidate proteins of interest were selected for label-free MS1 quantification using the Skyline software. MS1 full scan filtering and quantification of peptides identified hypoxia-induced surface proteins ( n =55) classified into the indicated groups. Data were obtained using the ConsensusPathDB interaction database. ( b ) Quantitative data of candidate proteins at the surface and following internalization. Shown is the distribution of proteins (% of total candidate proteins) according to fold change of protein expression in hypoxic versus normoxic HeLa cells (see also, Supplementary Data 1 , 2 , 3 and Supplementary Table 1 ). ( c ) Colour map of relative ratios of 32 candidate proteins at hypoxic versus normoxic conditions, isolated at the cell-surface and following internalization. The upper panel shows proteins with significant, hypoxic regulation of corresponding mRNAs. Gene expression data (right column) are presented as fold increase in hypoxic versus normoxic HeLa cells from three independent experiments. * P
    Figure Legend Snippet: Encoding of hypoxia-induced internalizing surface proteins. ( a ) HeLa cells were pre-treated at normoxia or hypoxia for 20 h. Total surface proteins and internalized proteins, following 2 h of endocytosis, were isolated by streptavidin affinity chromatography and identified by LC–MS/MS analysis. Candidate proteins of interest were selected for label-free MS1 quantification using the Skyline software. MS1 full scan filtering and quantification of peptides identified hypoxia-induced surface proteins ( n =55) classified into the indicated groups. Data were obtained using the ConsensusPathDB interaction database. ( b ) Quantitative data of candidate proteins at the surface and following internalization. Shown is the distribution of proteins (% of total candidate proteins) according to fold change of protein expression in hypoxic versus normoxic HeLa cells (see also, Supplementary Data 1 , 2 , 3 and Supplementary Table 1 ). ( c ) Colour map of relative ratios of 32 candidate proteins at hypoxic versus normoxic conditions, isolated at the cell-surface and following internalization. The upper panel shows proteins with significant, hypoxic regulation of corresponding mRNAs. Gene expression data (right column) are presented as fold increase in hypoxic versus normoxic HeLa cells from three independent experiments. * P

    Techniques Used: Isolation, Affinity Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Software, Expressing

    Hypoxia-induced CAIX overrides caveolin-1 negative regulation to allow specific cytotoxin delivery to hypoxic cells. ( a ) Left panel: HeLa cells pre-treated at normoxia or hypoxia for 20 h were analysed for CAIX by immunoblotting with tubulin as loading control. Right panel: HeLa cells were pre-treated as above, followed by cell-surface protein biotinylation and visualization of biotinylated CAIX. Negative control (Ctrl) represents non-biotinylated cells. ( b ) HeLa cells were surface biotinylated, followed by endocytosis for 30 min. Cells were stained for internalized proteins by streptavidin-AF-546 (magenta) and CAIX (green). Right panel shows CAIX co-localization with internalized proteins. Scale bar, 20 μm; right panel, 5 μm. ( c ) HeLa cells were pre-treated as in ( a ) and analysed by FACS for anti-CAIX antibody (α-CAIX) uptake at the indicated time points. ( d ) Caveolin-1 knockdown (Cav-1 KD) and control HeLa cells transduced with scrambled shRNA (Scr) were cultured at hypoxia for 20 h, and α-CAIX internalization and caveolin-1 expression were analysed by confocal microscopy. Scale bar, 20 μm. ( e ) FACS quantification from similar experiment as in ( d ). ( f ) Hypoxic Cav-1 KD cells transiently transfected with caveolin-1-expressing plasmid (pCav-1) were analysed for α-CAIX endocytosis, showing decreased internalization in caveolin-1 overexpressing cells (white dashed line). Scale bar, 20 μm. ( g ) Quantitative results from experiment in ( f ) using Cell Profiler, expressed as % relative to nontransfected Cav-1 KD cells±s.d. from six representative areas ( n =3). * P
    Figure Legend Snippet: Hypoxia-induced CAIX overrides caveolin-1 negative regulation to allow specific cytotoxin delivery to hypoxic cells. ( a ) Left panel: HeLa cells pre-treated at normoxia or hypoxia for 20 h were analysed for CAIX by immunoblotting with tubulin as loading control. Right panel: HeLa cells were pre-treated as above, followed by cell-surface protein biotinylation and visualization of biotinylated CAIX. Negative control (Ctrl) represents non-biotinylated cells. ( b ) HeLa cells were surface biotinylated, followed by endocytosis for 30 min. Cells were stained for internalized proteins by streptavidin-AF-546 (magenta) and CAIX (green). Right panel shows CAIX co-localization with internalized proteins. Scale bar, 20 μm; right panel, 5 μm. ( c ) HeLa cells were pre-treated as in ( a ) and analysed by FACS for anti-CAIX antibody (α-CAIX) uptake at the indicated time points. ( d ) Caveolin-1 knockdown (Cav-1 KD) and control HeLa cells transduced with scrambled shRNA (Scr) were cultured at hypoxia for 20 h, and α-CAIX internalization and caveolin-1 expression were analysed by confocal microscopy. Scale bar, 20 μm. ( e ) FACS quantification from similar experiment as in ( d ). ( f ) Hypoxic Cav-1 KD cells transiently transfected with caveolin-1-expressing plasmid (pCav-1) were analysed for α-CAIX endocytosis, showing decreased internalization in caveolin-1 overexpressing cells (white dashed line). Scale bar, 20 μm. ( g ) Quantitative results from experiment in ( f ) using Cell Profiler, expressed as % relative to nontransfected Cav-1 KD cells±s.d. from six representative areas ( n =3). * P

    Techniques Used: Negative Control, Staining, FACS, Transduction, shRNA, Cell Culture, Expressing, Confocal Microscopy, Transfection, Plasmid Preparation

    67) Product Images from "TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length"

    Article Title: TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112292

    FRET between streptavidin on plastic plates and IEkMCC tethered with PEG polymers. (A) Measured FRET efficiencies of IEkMCC tethered with six different PEG polymers. The intensity of DyLight 549 was captured before and after DyLight 649 was photobleached. The measured FRET efficiency ( ) was calculated using the intensity of DyLight 549 before ( ) and after ( ) DyLight 649 photobleaching ( ). The averaged values of two measurements were plotted with standard deviations. (B) After normalization, the measured FRET efficiencies match those calculated based on the Flory radius ( ) of the PEG polymers. The of the PEG polymer of subunits and unit length was calculated using , where is 0.28 nm [32] . Theoretical FRET efficiency ( ) was calculated using the equation , where the Förster distance ( ) of the DyLight 549-DyLight 649 donor-acceptor pair is 5 nm and the distance between the pMHC ligand and streptavidin is of the PEG polymer plus the pMHC radius of 2 nm. The FRET efficiencies were normalized by dividing the FRET efficiencies by the FRET efficiency of PEG 88.
    Figure Legend Snippet: FRET between streptavidin on plastic plates and IEkMCC tethered with PEG polymers. (A) Measured FRET efficiencies of IEkMCC tethered with six different PEG polymers. The intensity of DyLight 549 was captured before and after DyLight 649 was photobleached. The measured FRET efficiency ( ) was calculated using the intensity of DyLight 549 before ( ) and after ( ) DyLight 649 photobleaching ( ). The averaged values of two measurements were plotted with standard deviations. (B) After normalization, the measured FRET efficiencies match those calculated based on the Flory radius ( ) of the PEG polymers. The of the PEG polymer of subunits and unit length was calculated using , where is 0.28 nm [32] . Theoretical FRET efficiency ( ) was calculated using the equation , where the Förster distance ( ) of the DyLight 549-DyLight 649 donor-acceptor pair is 5 nm and the distance between the pMHC ligand and streptavidin is of the PEG polymer plus the pMHC radius of 2 nm. The FRET efficiencies were normalized by dividing the FRET efficiencies by the FRET efficiency of PEG 88.

    Techniques Used:

    Schematic illustration of IEkMCC ligands tethered onto a plastic surface with PEG polymer linkers. IEkMCC proteins with free c-terminal cysteines were first conjugated with heterobifunctional PEG linkers Mal-PEG-Bio through interactions between the sulfhydryl group and the maleimide group. Conjugates with biotin at the free ends of the polymer were then tethered to a plastic surface coated with streptavidin.
    Figure Legend Snippet: Schematic illustration of IEkMCC ligands tethered onto a plastic surface with PEG polymer linkers. IEkMCC proteins with free c-terminal cysteines were first conjugated with heterobifunctional PEG linkers Mal-PEG-Bio through interactions between the sulfhydryl group and the maleimide group. Conjugates with biotin at the free ends of the polymer were then tethered to a plastic surface coated with streptavidin.

    Techniques Used:

    68) Product Images from "Characterization for Binding Complex Formation with Site-Directly Immobilized Antibodies Enhancing Detection Capability of Cardiac Troponin I"

    Article Title: Characterization for Binding Complex Formation with Site-Directly Immobilized Antibodies Enhancing Detection Capability of Cardiac Troponin I

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2009/104094

    Comparison of the reactive antibody densities immobilized on the solid surfaces via biotin-streptavidin linkage. At a constant concentration of antibody, the reactive antibody density was determined by means of Scatchard analysis. The same procedure was repeated with different antibody preparations of IgG, F(ab′) 2 , and Fab. Standard deviations of triplicate repetition for each measurement were shown.
    Figure Legend Snippet: Comparison of the reactive antibody densities immobilized on the solid surfaces via biotin-streptavidin linkage. At a constant concentration of antibody, the reactive antibody density was determined by means of Scatchard analysis. The same procedure was repeated with different antibody preparations of IgG, F(ab′) 2 , and Fab. Standard deviations of triplicate repetition for each measurement were shown.

    Techniques Used: Concentration Assay

    Dose-response curves for binding between each type of biotinylated antibody and cTnI at low (0.5 pmol/mL addition) and high (2.5 pmol/mL) surface densities of the respective antibody. The antibodies were immobilized via biotin-streptavidin linkage and used as the capture for carrying out sandwich enzyme immunoassays for cTnI. The signals produced from the assays were plotted against the analyte concentration. The mean of triplicate determinations for each measurement and the corresponding standard deviations were indicated.
    Figure Legend Snippet: Dose-response curves for binding between each type of biotinylated antibody and cTnI at low (0.5 pmol/mL addition) and high (2.5 pmol/mL) surface densities of the respective antibody. The antibodies were immobilized via biotin-streptavidin linkage and used as the capture for carrying out sandwich enzyme immunoassays for cTnI. The signals produced from the assays were plotted against the analyte concentration. The mean of triplicate determinations for each measurement and the corresponding standard deviations were indicated.

    Techniques Used: Binding Assay, Enzyme Immunoassay, Produced, Concentration Assay

    69) Product Images from "Structural and Mechanistic Insight into DNA Unwinding by Deinococcus radiodurans UvrD"

    Article Title: Structural and Mechanistic Insight into DNA Unwinding by Deinococcus radiodurans UvrD

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077364

    ssDNA translocase activity of dr UvrD. Translocase activity of dr UvrD was assayed using the streptavidin-displacement assay. A. Structure of DNA oligonucleotides used for dr UvrD translocase assay measuring streptavidin displacement from biotinylated DNA substrates. The fluorescein label is represented as a star and the biotin label as a circle. B. Time course of dr UvrD (250 nM) catalyzed streptavidin displacement from the 3′- (blue) and 5′- (red) ssDNA extensions of DNA oligonucleotides shown in (A). The fraction of released dsDNA (no longer bound to streptavidin) was quantified and plotted as a function of time. C. Translocase activ ity of dr UvrD (250 nM) on 5' tailed dsDNA (20 nM) as a function of time in the absence (left) and the presence (right) of dr SSB (250 nM). The reaction products were analyzed on a 10 % polyacrylamide TBE gel. Bands correspond to the fluorescein labeled reaction products: streptavidin-bound dsDNA (upper bands, corresponding to several biotin labeled oligonucleotides bound to streptavidin), released dsDNA (middle band) and unwound ssDNA (lower band). D. The bands shown in (C), resulting from the time course of streptavidin displacement from 5′- tailed dsDNA, were quantified and the fraction of streptavidin-bound (black), released dsDNA (red) and unwound ssDNA (blue) were plotted as a function of time for reactions carried out in the absence (full lines) and presence (dotted lines) of dr SSB (250 nM). Standard deviations are shown as vertical bars.
    Figure Legend Snippet: ssDNA translocase activity of dr UvrD. Translocase activity of dr UvrD was assayed using the streptavidin-displacement assay. A. Structure of DNA oligonucleotides used for dr UvrD translocase assay measuring streptavidin displacement from biotinylated DNA substrates. The fluorescein label is represented as a star and the biotin label as a circle. B. Time course of dr UvrD (250 nM) catalyzed streptavidin displacement from the 3′- (blue) and 5′- (red) ssDNA extensions of DNA oligonucleotides shown in (A). The fraction of released dsDNA (no longer bound to streptavidin) was quantified and plotted as a function of time. C. Translocase activ ity of dr UvrD (250 nM) on 5' tailed dsDNA (20 nM) as a function of time in the absence (left) and the presence (right) of dr SSB (250 nM). The reaction products were analyzed on a 10 % polyacrylamide TBE gel. Bands correspond to the fluorescein labeled reaction products: streptavidin-bound dsDNA (upper bands, corresponding to several biotin labeled oligonucleotides bound to streptavidin), released dsDNA (middle band) and unwound ssDNA (lower band). D. The bands shown in (C), resulting from the time course of streptavidin displacement from 5′- tailed dsDNA, were quantified and the fraction of streptavidin-bound (black), released dsDNA (red) and unwound ssDNA (blue) were plotted as a function of time for reactions carried out in the absence (full lines) and presence (dotted lines) of dr SSB (250 nM). Standard deviations are shown as vertical bars.

    Techniques Used: Activity Assay, Labeling

    70) Product Images from "Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase"

    Article Title: Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase

    Journal: Viruses

    doi: 10.3390/v9060151

    Illustration of the polymerase assay set-up on the biosensor chip using DNA/RNA hybrid template. The biotinylated single-stranded DNA (ssDNA) was immobilized on chip via streptavidin/biotin interaction. Subsequently, a 31-mer RNA was hybridized to the DNA by 8 complementary base pairs. The polymerase activity was monitored in real-time by injecting NS5B with nucleotides over the immobilized DNA/RNA hybrid. S = streptavidin, B = biotin.
    Figure Legend Snippet: Illustration of the polymerase assay set-up on the biosensor chip using DNA/RNA hybrid template. The biotinylated single-stranded DNA (ssDNA) was immobilized on chip via streptavidin/biotin interaction. Subsequently, a 31-mer RNA was hybridized to the DNA by 8 complementary base pairs. The polymerase activity was monitored in real-time by injecting NS5B with nucleotides over the immobilized DNA/RNA hybrid. S = streptavidin, B = biotin.

    Techniques Used: Chromatin Immunoprecipitation, Activity Assay

    71) Product Images from "Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase"

    Article Title: Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase

    Journal: Viruses

    doi: 10.3390/v9060151

    Illustration of the polymerase assay set-up on the biosensor chip using DNA/RNA hybrid template. The biotinylated single-stranded DNA (ssDNA) was immobilized on chip via streptavidin/biotin interaction. Subsequently, a 31-mer RNA was hybridized to the DNA by 8 complementary base pairs. The polymerase activity was monitored in real-time by injecting NS5B with nucleotides over the immobilized DNA/RNA hybrid. S = streptavidin, B = biotin.
    Figure Legend Snippet: Illustration of the polymerase assay set-up on the biosensor chip using DNA/RNA hybrid template. The biotinylated single-stranded DNA (ssDNA) was immobilized on chip via streptavidin/biotin interaction. Subsequently, a 31-mer RNA was hybridized to the DNA by 8 complementary base pairs. The polymerase activity was monitored in real-time by injecting NS5B with nucleotides over the immobilized DNA/RNA hybrid. S = streptavidin, B = biotin.

    Techniques Used: Chromatin Immunoprecipitation, Activity Assay

    72) Product Images from "Ultrasound combined with targeted cationic microbubble-mediated angiogenesis gene transfection improves ischemic heart function"

    Article Title: Ultrasound combined with targeted cationic microbubble-mediated angiogenesis gene transfection improves ischemic heart function

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.4270

    (A) Schematic illustration of a TCMB. An ICAM-1 antibody was conjugated to the microbubbles using biotin-streptavidin bridging chemistry. (B) CMBs and TCMBs under light and fluorescence microscopy (scale bar, 15 µm). The green fluorescent ring around the microbubble surface indicates the combination of the fluorescein isothiocyanate-labeled immunoglobulin G and the ICAM-1 antibody on the surface of a cationic microbubble. ICAM-1, intercellular adhesion molecule-1; TCMB, targeted cationic microbubble; CMB, non-targeted cationic microbubbles; DSPE-PEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000].
    Figure Legend Snippet: (A) Schematic illustration of a TCMB. An ICAM-1 antibody was conjugated to the microbubbles using biotin-streptavidin bridging chemistry. (B) CMBs and TCMBs under light and fluorescence microscopy (scale bar, 15 µm). The green fluorescent ring around the microbubble surface indicates the combination of the fluorescein isothiocyanate-labeled immunoglobulin G and the ICAM-1 antibody on the surface of a cationic microbubble. ICAM-1, intercellular adhesion molecule-1; TCMB, targeted cationic microbubble; CMB, non-targeted cationic microbubbles; DSPE-PEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000].

    Techniques Used: Fluorescence, Microscopy, Labeling

    73) Product Images from "Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii"

    Article Title: Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s18072028

    ( A ) Schematic of the functionalization route for PEG-grafted gold nanoparticles and conjugation with anti- C. sakazakii IgG via streptavidin-biotin coupling; ( B ) the resulting immunogold nanoparticles specifically bind to C. sakazakii after incubation. A fast and simple centrifugation and filtration step makes the specifically labeled bacteria detectable with plasmon extinction spectroscopy.
    Figure Legend Snippet: ( A ) Schematic of the functionalization route for PEG-grafted gold nanoparticles and conjugation with anti- C. sakazakii IgG via streptavidin-biotin coupling; ( B ) the resulting immunogold nanoparticles specifically bind to C. sakazakii after incubation. A fast and simple centrifugation and filtration step makes the specifically labeled bacteria detectable with plasmon extinction spectroscopy.

    Techniques Used: Conjugation Assay, Incubation, Centrifugation, Filtration, Labeling, Spectroscopy

    Characterization of optical properties and protein-functionalization of Au nanoparticles grafted with HS-PEG-biotin. ( A ) UV-Vis extinction spectra of Au-core NPs grafted with PEG brushes and functionalized with different percentages of biotin end-groups binding to streptavidin and further binding biotinylated anti- C. sakazakii IgG suspended in 145.4 mM of NaCl; ( B ) detection of anti- C. sakazakii IgG bound to Au nanoparticles functionalized with different percentages of biotin end-groups and streptavidin by SDS-PAGE. Two controls of streptavidin: one set was mixed with non-reducing sample buffer (non-denaturing) and the other was mixed with reducing agent (denaturing). M: marker, STV*: non-reduced streptavidin, RIgG: standard Rabbit Immunoglobulin G from rabbit serum (commercial), STV: reduced streptavidin, black and blue arrows indicate the band of heavy and light chains of the antibody respectively; the red arrow indicates the band of streptavidin.
    Figure Legend Snippet: Characterization of optical properties and protein-functionalization of Au nanoparticles grafted with HS-PEG-biotin. ( A ) UV-Vis extinction spectra of Au-core NPs grafted with PEG brushes and functionalized with different percentages of biotin end-groups binding to streptavidin and further binding biotinylated anti- C. sakazakii IgG suspended in 145.4 mM of NaCl; ( B ) detection of anti- C. sakazakii IgG bound to Au nanoparticles functionalized with different percentages of biotin end-groups and streptavidin by SDS-PAGE. Two controls of streptavidin: one set was mixed with non-reducing sample buffer (non-denaturing) and the other was mixed with reducing agent (denaturing). M: marker, STV*: non-reduced streptavidin, RIgG: standard Rabbit Immunoglobulin G from rabbit serum (commercial), STV: reduced streptavidin, black and blue arrows indicate the band of heavy and light chains of the antibody respectively; the red arrow indicates the band of streptavidin.

    Techniques Used: Binding Assay, SDS Page, Marker

    UV-Vis extinction spectra of C. sakazakii incubated with nanoparticles. ( A ) C. sakazakii at a concentration of 10 3 CFU/mL incubated with targeted Au nanoparticles, functionalized with different percentages of biotin end-groups (HS-PEG-biotin 0–100%) and functionalized with biotinylated anti- C. sakazakii IgG via streptavidin. The samples were incubated for 2 h at room temperature and excess free particles thereafter were removed by filtration; ( B ) C. sakazakii at a concentration of 10 3 CFU/mL incubated with targeted Au nanoparticles stabilized with 100% HS-PEG-biotin and functionalized with biotinylated anti- C. sakazakii IgG via streptavidin. The sample was incubated for 2 h at room temperature and excess free particles thereafter were removed by filtration. The background of a sample containing only C. sakazakii was subtracted to reveal the extinction peak of the targeted Au nanoparticles. Also, the extinction peak of a pure dispersion of functionalized Au nanoparticles at known initial concentration with baseline correction; ( C ) negative controls of C. sakazakii and E. coli incubated with PEGylated Au NPs followed by removal of the free particles by filtration as well as C. sakazakii without Au NPs. Additionally, E. coli incubated with Au NPs 100% functionalized with anti- C. sakazakii , following the same procedure and conditions as in ( A ). ( D ) C. sakazakii at different concentrations 10 1 to 10 7 CFU/mL incubated with Au nanoparticles stabilized with 100% HS-PEG-biotin 100% and functionalized with biotinylated anti- C. sakazakii IgG via streptavidin. Please note that the sample containing 10 7 CFU/mL was diluted 25 times for the absorption measurement due to high background scattering.
    Figure Legend Snippet: UV-Vis extinction spectra of C. sakazakii incubated with nanoparticles. ( A ) C. sakazakii at a concentration of 10 3 CFU/mL incubated with targeted Au nanoparticles, functionalized with different percentages of biotin end-groups (HS-PEG-biotin 0–100%) and functionalized with biotinylated anti- C. sakazakii IgG via streptavidin. The samples were incubated for 2 h at room temperature and excess free particles thereafter were removed by filtration; ( B ) C. sakazakii at a concentration of 10 3 CFU/mL incubated with targeted Au nanoparticles stabilized with 100% HS-PEG-biotin and functionalized with biotinylated anti- C. sakazakii IgG via streptavidin. The sample was incubated for 2 h at room temperature and excess free particles thereafter were removed by filtration. The background of a sample containing only C. sakazakii was subtracted to reveal the extinction peak of the targeted Au nanoparticles. Also, the extinction peak of a pure dispersion of functionalized Au nanoparticles at known initial concentration with baseline correction; ( C ) negative controls of C. sakazakii and E. coli incubated with PEGylated Au NPs followed by removal of the free particles by filtration as well as C. sakazakii without Au NPs. Additionally, E. coli incubated with Au NPs 100% functionalized with anti- C. sakazakii , following the same procedure and conditions as in ( A ). ( D ) C. sakazakii at different concentrations 10 1 to 10 7 CFU/mL incubated with Au nanoparticles stabilized with 100% HS-PEG-biotin 100% and functionalized with biotinylated anti- C. sakazakii IgG via streptavidin. Please note that the sample containing 10 7 CFU/mL was diluted 25 times for the absorption measurement due to high background scattering.

    Techniques Used: Incubation, Concentration Assay, Filtration

    Transmission electron micrographs of C. sakazakii . ( A ) C. sakazakii ; ( B ) C. sakazakii incubated with PEG-grafted Au nanoparticles. The nanoparticles are only observed in the background; ( C ) two micrographs of C. sakazakii incubated with targeted Au nanoparticles stabilized with 100% HS-PEG-biotin and functionalized with biotinylated anti- C. sakazakii IgG via streptavidin. The samples were incubated for 2 h at room temperature and excess free particles thereafter were removed by filtration. The nanoparticles are found bound to the bacteria (identified by red arrows) in a manner consistent with binding to the cell membrane.
    Figure Legend Snippet: Transmission electron micrographs of C. sakazakii . ( A ) C. sakazakii ; ( B ) C. sakazakii incubated with PEG-grafted Au nanoparticles. The nanoparticles are only observed in the background; ( C ) two micrographs of C. sakazakii incubated with targeted Au nanoparticles stabilized with 100% HS-PEG-biotin and functionalized with biotinylated anti- C. sakazakii IgG via streptavidin. The samples were incubated for 2 h at room temperature and excess free particles thereafter were removed by filtration. The nanoparticles are found bound to the bacteria (identified by red arrows) in a manner consistent with binding to the cell membrane.

    Techniques Used: Transmission Assay, Incubation, Filtration, Binding Assay

    74) Product Images from "Anomalous Diffusion of Proteins Due to Molecular Crowding"

    Article Title: Anomalous Diffusion of Proteins Due to Molecular Crowding

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.104.051078

    ( a ) Anomalous diffusion exponent associated with the diffusion of streptavidin as a function of obstacle concentration for dextrans of various average molecular weights. Lines are fits to the data using Eq. 10 with α l = 0.74. Where necessary,
    Figure Legend Snippet: ( a ) Anomalous diffusion exponent associated with the diffusion of streptavidin as a function of obstacle concentration for dextrans of various average molecular weights. Lines are fits to the data using Eq. 10 with α l = 0.74. Where necessary,

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Effective distributions in average residence times calculated with the MEMFCS algorithm for the experimental autocorrelation data shown in ( symbols ), and for two sets of simulated autocorrelation data corresponding to the case of streptavidin
    Figure Legend Snippet: Effective distributions in average residence times calculated with the MEMFCS algorithm for the experimental autocorrelation data shown in ( symbols ), and for two sets of simulated autocorrelation data corresponding to the case of streptavidin

    Techniques Used:

    Anomalous diffusion exponent as a function of dextran concentration fitted to Eq. 10 for various tracers: EGFP and streptavidin in solutions crowded with the 276.5 kDa dextran, and fluorescein and 282 kDa FITC-dextran in solutions crowded with 401.3 kDa
    Figure Legend Snippet: Anomalous diffusion exponent as a function of dextran concentration fitted to Eq. 10 for various tracers: EGFP and streptavidin in solutions crowded with the 276.5 kDa dextran, and fluorescein and 282 kDa FITC-dextran in solutions crowded with 401.3 kDa

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Anomalous diffusion exponents associated with the diffusion of streptavidin in solutions crowded with either BSA or nonfluorescent streptavidin for different concentrations of the obstacle proteins fitted to Eq. 10. For comparison, the exponent associated
    Figure Legend Snippet: Anomalous diffusion exponents associated with the diffusion of streptavidin in solutions crowded with either BSA or nonfluorescent streptavidin for different concentrations of the obstacle proteins fitted to Eq. 10. For comparison, the exponent associated

    Techniques Used: Diffusion-based Assay

    ( a ) Apparent diffusion coefficient, D ( τ D ), associated with the diffusion of streptavidin as a function of dextran concentration for dextrans of various average molecular weights ( open and solid symbols ). Also shown is D ( τ D ) for a 282 kDa
    Figure Legend Snippet: ( a ) Apparent diffusion coefficient, D ( τ D ), associated with the diffusion of streptavidin as a function of dextran concentration for dextrans of various average molecular weights ( open and solid symbols ). Also shown is D ( τ D ) for a 282 kDa

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Anomalous diffusion exponent α associated with the diffusion of streptavidin in presence of 75 g/l and 200 g/l of 276 kDa dextran as a function of the temperature. The average values of the anomalous diffusion exponent α over the considered
    Figure Legend Snippet: Anomalous diffusion exponent α associated with the diffusion of streptavidin in presence of 75 g/l and 200 g/l of 276 kDa dextran as a function of the temperature. The average values of the anomalous diffusion exponent α over the considered

    Techniques Used: Diffusion-based Assay

    Anomalous diffusion exponent corresponding to the diffusion of streptavidin in PBS with and without 200 g/l of 276 kDa dextran, as a function of added NaCl.
    Figure Legend Snippet: Anomalous diffusion exponent corresponding to the diffusion of streptavidin in PBS with and without 200 g/l of 276 kDa dextran, as a function of added NaCl.

    Techniques Used: Diffusion-based Assay

    75) Product Images from "Mitochondria distinguish granule-stored from de novo synthesized TNF secretion in human mast cells"

    Article Title: Mitochondria distinguish granule-stored from de novo synthesized TNF secretion in human mast cells

    Journal: International archives of allergy and immunology

    doi: 10.1159/000335178

    IgE/ streptavidin -triggered hCBMCs degranualtion and TNF secretion is associated with mitochondrial translocation
    Figure Legend Snippet: IgE/ streptavidin -triggered hCBMCs degranualtion and TNF secretion is associated with mitochondrial translocation

    Techniques Used: Translocation Assay

    Related Articles

    Blocking Assay:

    Article Title: Quality control for unfolded proteins at the plasma membrane
    Article Snippet: CD4 chimeras were internalized for 40 min at 37°C, and the remaining PM biotin-Fab was blocked by 10 µg/ml streptavidin at 0°C (Sigma-Aldrich). .. The nonspecific Ab background as well as the residual signal derived after streptavidin blocking was taken into account when calculating the chimera recycling efficiency.

    Electrophoresis:

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Samples of the reaction products were added to an equal volume of loading buffer [100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue] and analysed by native gel electrophoresis using 12% (w/v) 29:1 acrylamide : bis -acrylamide gels containing 150 mM Tris-HCl, pH 6.8 in the upper stacking gel and 375 mM Tris-HCl, pH 8.8 in the resolving gel and electrophoresis buffer containing 192 mM glycine and 25 mM Tris-HCl, pH 8.3.

    Incubation:

    Article Title: Atomic Force Microscope Imaging of Chromatin Assembled in Xenopus laevis Egg Extract
    Article Snippet: .. The streptavidin coated surface was prepared by the following standard procedure: 1) cleaned glass or mica surface was first sinalized by APTES (Sigma) followed by coating with glutaraldehyde (Sigma), 2) 0.02 mg/ml streptavidin (Sigma) in 1x phosphate buffered saline (PBS) was incubated on the above surface for eight hours at 24°C, 3) the streptavidin coated surface was passivated with 0.5 M etholamine (Sigma) for one hour at 24°C and then stored in 10 mg/ml bovine serum albumin (BSA) (Sigma) at 4°C. ..

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: .. Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Samples of the reaction products were added to an equal volume of loading buffer [100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue] and analysed by native gel electrophoresis using 12% (w/v) 29:1 acrylamide : bis -acrylamide gels containing 150 mM Tris-HCl, pH 6.8 in the upper stacking gel and 375 mM Tris-HCl, pH 8.8 in the resolving gel and electrophoresis buffer containing 192 mM glycine and 25 mM Tris-HCl, pH 8.3.

    Article Title: A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
    Article Snippet: .. If the detection of biotin-dUTP was performed by fluorescently labeled streptavidin, the samples were incubated with streptavidin from Streptomyces avidinii conjugated with FITC (1:100, Sigma Aldrich, Prague, Czech Republic) diluted in the Tris-NaCl buffer for 30 min. After washing with the Tris-NaCl buffer, the samples were mounted in the mounting medium. .. In some experiments, the mitochondrial marker MTCO2 (1:100, Abcam, Cambridge, United Kingdom) was added to the mixture of the primary antibody.

    Article Title: Upstream Stimulatory Factors 1 and 2 Mediate the Transcription of Angiotensin II Binding and Inhibitory Protein *
    Article Snippet: .. The streptavidin that was immobilized on agarose CL-4B (85881, Sigma) was pretreated with TN buffer containing 1% BSA and incubated with 50 μg of nuclear extracts from mDCT cells on ice in a 200-μl EMSA binding buffer for 20 min. After five washing steps with EMSA binding buffer, the streptavidin-biotin-DNA complex was eluted with SDS buffer, and a one-fifth volume was used for immunoblot analysis. .. Chromatin Immunoprecipitation (ChIP) Assay ChIP assay was performed essentially according to the manufacturer's protocol (Active Motif) ( , ).

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: The binding studies with the biotinylated DNA were performed by sequential incubation of 168 ng of the biotinylated DNA in the presence of 7.2 mM CaCl2 with 4.7 µg Csn2, 14 mM ethylene glycol tetraacetic acid (EGTA) and/or 2 µg streptavidin (Sigma-Aldrich) in a total volume of 14.4 µl. .. The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore).

    Derivative Assay:

    Article Title: Quality control for unfolded proteins at the plasma membrane
    Article Snippet: CD4 chimeras were internalized for 40 min at 37°C, and the remaining PM biotin-Fab was blocked by 10 µg/ml streptavidin at 0°C (Sigma-Aldrich). .. The nonspecific Ab background as well as the residual signal derived after streptavidin blocking was taken into account when calculating the chimera recycling efficiency.

    Conjugation Assay:

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: .. Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Samples of the reaction products were added to an equal volume of loading buffer [100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue] and analysed by native gel electrophoresis using 12% (w/v) 29:1 acrylamide : bis -acrylamide gels containing 150 mM Tris-HCl, pH 6.8 in the upper stacking gel and 375 mM Tris-HCl, pH 8.8 in the resolving gel and electrophoresis buffer containing 192 mM glycine and 25 mM Tris-HCl, pH 8.3.

    Chromatography:

    Article Title: Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin
    Article Snippet: Periodate oxidation of avidin and other glycoproteins Avidin (Tecnogen) was oxidized with 20mM sodium periodate (NaIO4 ) (Sigma- Aldrich), in 100 mM acetate at pH 5.5 for 1 hour at room temperature, in the presence of a molar excess of 4-hydroxyazobenzene-2′-carboxylic acid (HABA) (Sigma-Aldrich) and purified by chromatography on a Sephadex G-25 fine desalting column (GE Healthcare) as previously reported . .. Glycosylated streptavidin was obtained by reacting streptavidin with α-D-mannopyranosylphenylisothiocyanate (Sigma-Aldrich) at 1∶25 molar ratio at +4°C for 5 or 24 hours and purified by size exclusion chromatography.

    Imaging:

    Article Title: Atomic Force Microscope Imaging of Chromatin Assembled in Xenopus laevis Egg Extract
    Article Snippet: Paragraph title: AFM Imaging ... The streptavidin coated surface was prepared by the following standard procedure: 1) cleaned glass or mica surface was first sinalized by APTES (Sigma) followed by coating with glutaraldehyde (Sigma), 2) 0.02 mg/ml streptavidin (Sigma) in 1x phosphate buffered saline (PBS) was incubated on the above surface for eight hours at 24°C, 3) the streptavidin coated surface was passivated with 0.5 M etholamine (Sigma) for one hour at 24°C and then stored in 10 mg/ml bovine serum albumin (BSA) (Sigma) at 4°C.

    Polymerase Chain Reaction:

    Article Title: Liposomes on a streptavidin crystal: a system to study membrane proteins by cryo-EM
    Article Snippet: Streptavidin was purchased from Sigma-Aldrich (St Louis, MO). .. 67 μl of Tris buffer (150 mM NaCl, 50 mM Tris, pH 7.0) containing 0.05-0.40 mg/ml of streptavidin is deposited in a micro-well formed by the polypropylene cap of a 0.2 ml PCR tube.

    Binding Assay:

    Article Title: Electrolyte-free Amperometric Immunosensor using a Dendritic Nanotip
    Article Snippet: .. Streptavidin (1mg/mL in 1xPBS, Sigma) was nonspecifically immobilized on the surface followed by the binding of biotinylated polyclonal IgY antibodies (5 mg/ml biotin-Antibody in PBS). ..

    Article Title: Upstream Stimulatory Factors 1 and 2 Mediate the Transcription of Angiotensin II Binding and Inhibitory Protein *
    Article Snippet: .. The streptavidin that was immobilized on agarose CL-4B (85881, Sigma) was pretreated with TN buffer containing 1% BSA and incubated with 50 μg of nuclear extracts from mDCT cells on ice in a 200-μl EMSA binding buffer for 20 min. After five washing steps with EMSA binding buffer, the streptavidin-biotin-DNA complex was eluted with SDS buffer, and a one-fifth volume was used for immunoblot analysis. .. Chromatin Immunoprecipitation (ChIP) Assay ChIP assay was performed essentially according to the manufacturer's protocol (Active Motif) ( , ).

    Article Title: Quality control for unfolded proteins at the plasma membrane
    Article Snippet: The Ab binding and detection were performed as described in the Cell surface density measurements of CD4 chimeras and GPCRs section. .. CD4 chimeras were internalized for 40 min at 37°C, and the remaining PM biotin-Fab was blocked by 10 µg/ml streptavidin at 0°C (Sigma-Aldrich).

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: .. The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore). .. The complexes were separated from free DNA on a native 2% Tris/Acetate agarose gel.

    Article Title: Liposomes on a streptavidin crystal: a system to study membrane proteins by cryo-EM
    Article Snippet: In the resulting crystal, each streptavidin tetramer has two vacant biotin binding sites that are available to bind to other molecules. .. Streptavidin was purchased from Sigma-Aldrich (St Louis, MO).

    Immunofluorescence:

    Article Title: A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
    Article Snippet: In the case of indirect immunofluorescence, the samples were incubated with the rabbit anti-biotin antibody (1:100, Abcam, Cambridge, United Kingdom) or mouse anti-digoxigenin antibody (1:100, Roche, primary antibodies) diluted in a Tris-NaCl buffer for 30 min, washed three times with the Tris-NaCl buffer, and then incubated with the anti-rabbit or anti-mouse antibody conjugated with the fluorochrome Alexa Fluor 488 diluted in the Tris-NaCl buffer (1:100, Jackson ImmunoResearch, secondary antibodies) for 30 min. .. If the detection of biotin-dUTP was performed by fluorescently labeled streptavidin, the samples were incubated with streptavidin from Streptomyces avidinii conjugated with FITC (1:100, Sigma Aldrich, Prague, Czech Republic) diluted in the Tris-NaCl buffer for 30 min. After washing with the Tris-NaCl buffer, the samples were mounted in the mounting medium.

    Nucleic Acid Electrophoresis:

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Samples of the reaction products were added to an equal volume of loading buffer [100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue] and analysed by native gel electrophoresis using 12% (w/v) 29:1 acrylamide : bis -acrylamide gels containing 150 mM Tris-HCl, pH 6.8 in the upper stacking gel and 375 mM Tris-HCl, pH 8.8 in the resolving gel and electrophoresis buffer containing 192 mM glycine and 25 mM Tris-HCl, pH 8.3.

    Mutagenesis:

    Article Title: pH-Dependent Deformations of the Energy Landscape of Avidin-like Proteins Investigated by Single Molecule Force Spectroscopy
    Article Snippet: Also the Avidin and Streptavidin are commercially available and can be brought from Sigma-Aldrich. .. Chimericavidin is not purchasable, we got this protein mutant from our partner in Finland, Vesa Hytönen and Markuu Kulomaa from the University of Tampere.

    Avidin-Biotin Assay:

    Article Title: Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin
    Article Snippet: Paragraph title: Periodate oxidation of avidin and other glycoproteins ... Glycosylated streptavidin was obtained by reacting streptavidin with α-D-mannopyranosylphenylisothiocyanate (Sigma-Aldrich) at 1∶25 molar ratio at +4°C for 5 or 24 hours and purified by size exclusion chromatography.

    Article Title: pH-Dependent Deformations of the Energy Landscape of Avidin-like Proteins Investigated by Single Molecule Force Spectroscopy
    Article Snippet: .. Also the Avidin and Streptavidin are commercially available and can be brought from Sigma-Aldrich. .. Chimericavidin is not purchasable, we got this protein mutant from our partner in Finland, Vesa Hytönen and Markuu Kulomaa from the University of Tampere.

    Labeling:

    Article Title: A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
    Article Snippet: .. If the detection of biotin-dUTP was performed by fluorescently labeled streptavidin, the samples were incubated with streptavidin from Streptomyces avidinii conjugated with FITC (1:100, Sigma Aldrich, Prague, Czech Republic) diluted in the Tris-NaCl buffer for 30 min. After washing with the Tris-NaCl buffer, the samples were mounted in the mounting medium. .. In some experiments, the mitochondrial marker MTCO2 (1:100, Abcam, Cambridge, United Kingdom) was added to the mixture of the primary antibody.

    Article Title: Quality control for unfolded proteins at the plasma membrane
    Article Snippet: To follow recycling, the surface-bound anti-CD4 Ab (1:1,000; BD) was labeled with biotin-conjugated anti–mouse Fab (216–1,806; KPL, Inc.) for 1 h on ice. .. CD4 chimeras were internalized for 40 min at 37°C, and the remaining PM biotin-Fab was blocked by 10 µg/ml streptavidin at 0°C (Sigma-Aldrich).

    Purification:

    Article Title: Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin
    Article Snippet: .. Glycosylated streptavidin was obtained by reacting streptavidin with α-D-mannopyranosylphenylisothiocyanate (Sigma-Aldrich) at 1∶25 molar ratio at +4°C for 5 or 24 hours and purified by size exclusion chromatography. ..

    Article Title: Differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization
    Article Snippet: Materials Chemokines and mutants were recombinantly expressed and purified as described previously [ , , ]. .. Streptavidin and fluorescently labelled streptavidin (Sigma Aldrich), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine-CAP-biotin (DOPE-CAP-biotin) (Avanti Polar Lipids, Alabaster, AL, USA) were purchased.

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: After purification of the DNA fragments by agarose gel electrophoresis, the biotinylation of the ends was achieved by Klenow incorporation of Biotin-11-dUTP. .. The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore).

    Marker:

    Article Title: A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
    Article Snippet: If the detection of biotin-dUTP was performed by fluorescently labeled streptavidin, the samples were incubated with streptavidin from Streptomyces avidinii conjugated with FITC (1:100, Sigma Aldrich, Prague, Czech Republic) diluted in the Tris-NaCl buffer for 30 min. After washing with the Tris-NaCl buffer, the samples were mounted in the mounting medium. .. In some experiments, the mitochondrial marker MTCO2 (1:100, Abcam, Cambridge, United Kingdom) was added to the mixture of the primary antibody.

    Titration:

    Article Title: Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin
    Article Snippet: Glycosylated streptavidin was obtained by reacting streptavidin with α-D-mannopyranosylphenylisothiocyanate (Sigma-Aldrich) at 1∶25 molar ratio at +4°C for 5 or 24 hours and purified by size exclusion chromatography. .. Avidin acetylation was performed by reaction with acetic acid N-hydroxysuccinimide ester (Sigma-Aldrich) according to a described method and the extent of amino group acetylation was determined by titration with TNBS as before.

    Plasmid Preparation:

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: Biotinylation of dsDNA fragment and Csn2 binding studies The 256 bp DNA fragment was obtained by Eco RI-Bam HI digestion of the plasmid pUC18-1. .. The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore).

    Agarose Gel Electrophoresis:

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: After purification of the DNA fragments by agarose gel electrophoresis, the biotinylation of the ends was achieved by Klenow incorporation of Biotin-11-dUTP. .. The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore).

    Size-exclusion Chromatography:

    Article Title: Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin
    Article Snippet: .. Glycosylated streptavidin was obtained by reacting streptavidin with α-D-mannopyranosylphenylisothiocyanate (Sigma-Aldrich) at 1∶25 molar ratio at +4°C for 5 or 24 hours and purified by size exclusion chromatography. ..

    Ethanol Precipitation:

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: After extraction with phenol/chloroform and ethanol precipitation, the DNA was dissolved in 10 µl TE buffer. .. The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore).

    Concentration Assay:

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Products from the reaction with the lowest concentration of 5′-biotinylated RNA that shifted all of the streptavidin were used in subsequent experiments.

    Article Title: A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy
    Article Snippet: If the detection of biotin-dUTP was performed by fluorescently labeled streptavidin, the samples were incubated with streptavidin from Streptomyces avidinii conjugated with FITC (1:100, Sigma Aldrich, Prague, Czech Republic) diluted in the Tris-NaCl buffer for 30 min. After washing with the Tris-NaCl buffer, the samples were mounted in the mounting medium. .. If DAPI was used, it was added to the mixture of the secondary antibody (the final concentration was 0.5 µg/mL).

    Article Title: Peptide Functionalization of Gold Nanoparticles for the Detection of Carcinoembryonic Antigen in Blood Plasma via SPR-Based Biosensor
    Article Snippet: Sodium acetate buffer solution 3M, pH 5.2 (25°C), KH2 PO4 , Na2 HPO4 , KCl, NaCl, ethanolamine, bovine serum albumin (BSA), streptavidin, and glutaraldehyde are purchased in molecular biology grade or higher from Sigma-Aldrich, USA. .. PBSNaCl is obtained, raising the NaCl concentration of the phosphate buffer to 750 mM.

    Standard Deviation:

    Article Title: Atomic Force Microscope Imaging of Chromatin Assembled in Xenopus laevis Egg Extract
    Article Snippet: Finally, more than 100 line-scans for the higher order chromatins obtained in the direct AFM imaging method under fixed condition were used to measure the width and standard deviation. .. The streptavidin coated surface was prepared by the following standard procedure: 1) cleaned glass or mica surface was first sinalized by APTES (Sigma) followed by coating with glutaraldehyde (Sigma), 2) 0.02 mg/ml streptavidin (Sigma) in 1x phosphate buffered saline (PBS) was incubated on the above surface for eight hours at 24°C, 3) the streptavidin coated surface was passivated with 0.5 M etholamine (Sigma) for one hour at 24°C and then stored in 10 mg/ml bovine serum albumin (BSA) (Sigma) at 4°C.

    Staining:

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Gels were stained using 0.2% (w/v) Coomassie Brilliant Blue R in 50% (v/v) methanol and 7% (v/v) glacial acetic acid and proteins visualized following destaining with 20% (v/v) methanol and 7% (v/v) glacial acetic acid.

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore). .. The bands were visualized by ethidium bromide staining.

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  • 99
    Millipore streptavidin
    ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of <t>streptavidin</t> (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).
    Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin/product/Millipore
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    86
    Millipore irdye800cw streptavidin overlay
    Crosslinking with BioATP-HDZ. ( A ) Structure of 2-azidoadenosine 5′-trisphosphate 2′,3′-biotin-long-chain-hydrazone (BioATP-HDZ). A highly reactive nitrene is produced by UV exposure and can form a covalent bond with a neighboring peptide backbone or amino acid side chain of the protein. ( B ) Fast-twitch skeletal muscle SR membranes were specifically labeled by 10 µM BioATP-HDZ and detected by <t>IRDye800CW-streptavidin</t> in-gel overlay. The left panel is the streptavidin in-gel overlay and the right panel is the CBB stain of the same gel. Lane 1 – non-labeled SR membranes; lane 2 – SR membranes labeled with BioATP-HDZ; lane 3 – competition of BioATP-HDZ labeling by ATP in SR membranes. ( C ) Western blot with anti-RyR1 (pseudo-colored red) and anti-SERCA2 (pseudo-colored green) antibodies.
    Irdye800cw Streptavidin Overlay, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irdye800cw streptavidin overlay/product/Millipore
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    88
    Millipore streptavidin sepharose affinity columns
    <t>Streptavidin-affinity-enriched</t> PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using <t>streptavidin—Sepharose</t> affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.
    Streptavidin Sepharose Affinity Columns, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore phycoerythrin coupled streptavidin
    Internal standard curves were constructed from four synthetic peptide standards in each well of a 96-well plate. The mean fluorescence intensity from four standards with varying degrees of immobilized synthetically phosphorylated Abltide is best modeled by a Boltzmann sigmoidal curve. Identical sets of standards labeled with either PY20-PE ( A ) or biotinylated 4G10 and <t>streptavidin-PE</t> ( B ) displayed large differences in the maximum fluorescence intensity. Within a single 96-well plate, well-specific standard curves displayed differences in slope and in the percentage of phosphorylation resulting in half maximal fluorescence (V 50 ), as calculated from the average of 100-300 measurements per standard per well. Serial dilutions of fluorescent anti-phosphotyrosine antibodies were used to confirm the effect of concentration on maximal fluorescence and slope. C , PY20-PE at the following concentrations in decreasing order: 150, 15, 1.5, 0.15 ng/mL. D , 4G10-biotin at the following concentrations in decreasing order: 1000, 100, 10, 1 ng/mL. Error bars for all mean fluorescence measurements indicate 99% confidence intervals.
    Phycoerythrin Coupled Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phycoerythrin coupled streptavidin/product/Millipore
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    ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of streptavidin (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).

    Journal: Nucleic Acids Research

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

    doi: 10.1093/nar/gkt315

    Figure Lengend Snippet: ( A ) Binding analyses of Csn2 in the presence and absence of EGTA and free DNA ends on 2% Tris-acetate agarose gel. In each lane 168 ng linear DNA and 7.2 mM CaCl 2 were employed. The numbers above the lanes indicate the order of addition of streptavidin (2 µg), Csn2 (4.7 µg), or EGTA (14 mM) in a total volume of 14.4 µl. Lanes 2–5: Influence of EGTA on Csn2-DNA interaction is shown. Lanes 6–9: 168 ng of the end-biotinylated DNA fragment were incubated first with streptavidin to block the DNA ends. Lanes 10 and 11: Streptavidin was added after binding of Csn2. After separation of the complexes the agarose gel was stained with ethidium bromide. ( B ) Schematic presentation of the binding analysis, shown in (A).

    Article Snippet: The volumes of the binding reaction without EGTA or streptavidin were adjusted by addition of deionized water (Millipore).

    Techniques: Binding Assay, Agarose Gel Electrophoresis, Incubation, Blocking Assay, Staining

    Crosslinking with BioATP-HDZ. ( A ) Structure of 2-azidoadenosine 5′-trisphosphate 2′,3′-biotin-long-chain-hydrazone (BioATP-HDZ). A highly reactive nitrene is produced by UV exposure and can form a covalent bond with a neighboring peptide backbone or amino acid side chain of the protein. ( B ) Fast-twitch skeletal muscle SR membranes were specifically labeled by 10 µM BioATP-HDZ and detected by IRDye800CW-streptavidin in-gel overlay. The left panel is the streptavidin in-gel overlay and the right panel is the CBB stain of the same gel. Lane 1 – non-labeled SR membranes; lane 2 – SR membranes labeled with BioATP-HDZ; lane 3 – competition of BioATP-HDZ labeling by ATP in SR membranes. ( C ) Western blot with anti-RyR1 (pseudo-colored red) and anti-SERCA2 (pseudo-colored green) antibodies.

    Journal: PLoS ONE

    Article Title: Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

    doi: 10.1371/journal.pone.0048725

    Figure Lengend Snippet: Crosslinking with BioATP-HDZ. ( A ) Structure of 2-azidoadenosine 5′-trisphosphate 2′,3′-biotin-long-chain-hydrazone (BioATP-HDZ). A highly reactive nitrene is produced by UV exposure and can form a covalent bond with a neighboring peptide backbone or amino acid side chain of the protein. ( B ) Fast-twitch skeletal muscle SR membranes were specifically labeled by 10 µM BioATP-HDZ and detected by IRDye800CW-streptavidin in-gel overlay. The left panel is the streptavidin in-gel overlay and the right panel is the CBB stain of the same gel. Lane 1 – non-labeled SR membranes; lane 2 – SR membranes labeled with BioATP-HDZ; lane 3 – competition of BioATP-HDZ labeling by ATP in SR membranes. ( C ) Western blot with anti-RyR1 (pseudo-colored red) and anti-SERCA2 (pseudo-colored green) antibodies.

    Article Snippet: Visualization of the photoaffinity labeled polypeptides was performed by IRDye800CW-streptavidin overlay either in-gel or after an over-night transfer to Immobilon-FL (Millipore).

    Techniques: Produced, Labeling, Staining, Western Blot

    Quantification of the binding affinity of BioATP-HDZ to RyR1. SR membranes were labeled with 10 µM BioATP-HDZ in the absence or presence of increasing concentrations of ATP. The crosslinking of BioATP-HDZ to RyR1 was determined by IRDye800CW-streptavidin in-gel overlay (pseudo-colored green, A ), and the signal was normalized to its respective CBB stain intensity at 700 nm (pseudo-colored red, B ). ( C ) Quantification of BioATP-HDZ crosslinking to RyR1 as the mean of 3 independent experiments ± SEM. The IC 50 determined by non-linear regression was 0.6±0.2 mM.

    Journal: PLoS ONE

    Article Title: Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

    doi: 10.1371/journal.pone.0048725

    Figure Lengend Snippet: Quantification of the binding affinity of BioATP-HDZ to RyR1. SR membranes were labeled with 10 µM BioATP-HDZ in the absence or presence of increasing concentrations of ATP. The crosslinking of BioATP-HDZ to RyR1 was determined by IRDye800CW-streptavidin in-gel overlay (pseudo-colored green, A ), and the signal was normalized to its respective CBB stain intensity at 700 nm (pseudo-colored red, B ). ( C ) Quantification of BioATP-HDZ crosslinking to RyR1 as the mean of 3 independent experiments ± SEM. The IC 50 determined by non-linear regression was 0.6±0.2 mM.

    Article Snippet: Visualization of the photoaffinity labeled polypeptides was performed by IRDye800CW-streptavidin overlay either in-gel or after an over-night transfer to Immobilon-FL (Millipore).

    Techniques: Binding Assay, Labeling, Staining

    Detection of BioATP-HDZ-labeled tryptic fragments. SR membranes were labeled with BioATP-HDZ in the absence ( A , B ) and presence ( C , D ) of ATP. Following the crosslinking, the SR membranes were digested with trypsin, solubilized with 2% CHAPS and separated by sucrose gradient centrifugation. Sucrose gradient fractions were analyzed by SDS-PAGE and labeled fragments were detected by in-gel IRDye800CW-streptavidin overlay at 800 nm. ( A , C ). The gels were then stained with CBB and scanned at 700 nm ( B , D ). Sucrose gradient RyR1 peak fractions are shown; fraction 20 was analyzed as an internal control for BioATP-HDZ labeling of SERCA. The number of the sucrose gradient fraction run in each lane is labeled above gels in A–D. ( E ) Purified RyR1 crosslinked with BioATP-HDZ, digested with trypsin and transferred to immobilon-FL membrane: lane 1 – molecular weight standards; lane 2 – IRDye800CW-streptavidin overlay; lane 3 – CBB staining. ( F ) Immunoblotting of the trypsin-digested labeled RyR1 with a specific antibody against RyR1 amino acid sequence 416–434. ( G ) Plot of the normalized relative fluorescence (RFU) calculated for individual BioATP-HDZ-labeled tryptic fragments of RyR1 detected in SR membranes (blue) and purified RyR1 (red), error bars represent SEM (N = 3) and * indicates p

    Journal: PLoS ONE

    Article Title: Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

    doi: 10.1371/journal.pone.0048725

    Figure Lengend Snippet: Detection of BioATP-HDZ-labeled tryptic fragments. SR membranes were labeled with BioATP-HDZ in the absence ( A , B ) and presence ( C , D ) of ATP. Following the crosslinking, the SR membranes were digested with trypsin, solubilized with 2% CHAPS and separated by sucrose gradient centrifugation. Sucrose gradient fractions were analyzed by SDS-PAGE and labeled fragments were detected by in-gel IRDye800CW-streptavidin overlay at 800 nm. ( A , C ). The gels were then stained with CBB and scanned at 700 nm ( B , D ). Sucrose gradient RyR1 peak fractions are shown; fraction 20 was analyzed as an internal control for BioATP-HDZ labeling of SERCA. The number of the sucrose gradient fraction run in each lane is labeled above gels in A–D. ( E ) Purified RyR1 crosslinked with BioATP-HDZ, digested with trypsin and transferred to immobilon-FL membrane: lane 1 – molecular weight standards; lane 2 – IRDye800CW-streptavidin overlay; lane 3 – CBB staining. ( F ) Immunoblotting of the trypsin-digested labeled RyR1 with a specific antibody against RyR1 amino acid sequence 416–434. ( G ) Plot of the normalized relative fluorescence (RFU) calculated for individual BioATP-HDZ-labeled tryptic fragments of RyR1 detected in SR membranes (blue) and purified RyR1 (red), error bars represent SEM (N = 3) and * indicates p

    Article Snippet: Visualization of the photoaffinity labeled polypeptides was performed by IRDye800CW-streptavidin overlay either in-gel or after an over-night transfer to Immobilon-FL (Millipore).

    Techniques: Labeling, Gradient Centrifugation, SDS Page, Staining, Purification, Molecular Weight, Sequencing, Fluorescence

    Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Journal: PLoS ONE

    Article Title: Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

    doi: 10.1371/journal.pone.0133033

    Figure Lengend Snippet: Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Article Snippet: Protein fractions were eluted by streptavidin—Sepharose affinity columns, desalted, concentrated using AmiconTM Ultrafree centrifugal filters (Millipore), and visualized using a FOCUS-FAST silver-stain kit.

    Techniques: Affinity Chromatography, Far Western Blot, Silver Staining

    Internal standard curves were constructed from four synthetic peptide standards in each well of a 96-well plate. The mean fluorescence intensity from four standards with varying degrees of immobilized synthetically phosphorylated Abltide is best modeled by a Boltzmann sigmoidal curve. Identical sets of standards labeled with either PY20-PE ( A ) or biotinylated 4G10 and streptavidin-PE ( B ) displayed large differences in the maximum fluorescence intensity. Within a single 96-well plate, well-specific standard curves displayed differences in slope and in the percentage of phosphorylation resulting in half maximal fluorescence (V 50 ), as calculated from the average of 100-300 measurements per standard per well. Serial dilutions of fluorescent anti-phosphotyrosine antibodies were used to confirm the effect of concentration on maximal fluorescence and slope. C , PY20-PE at the following concentrations in decreasing order: 150, 15, 1.5, 0.15 ng/mL. D , 4G10-biotin at the following concentrations in decreasing order: 1000, 100, 10, 1 ng/mL. Error bars for all mean fluorescence measurements indicate 99% confidence intervals.

    Journal: Molecular cancer therapeutics

    Article Title: A bead-based activity screen for small-molecule inhibitors of signal transduction in chronic myelogenous leukemia cells

    doi: 10.1158/1535-7163.MCT-10-0157

    Figure Lengend Snippet: Internal standard curves were constructed from four synthetic peptide standards in each well of a 96-well plate. The mean fluorescence intensity from four standards with varying degrees of immobilized synthetically phosphorylated Abltide is best modeled by a Boltzmann sigmoidal curve. Identical sets of standards labeled with either PY20-PE ( A ) or biotinylated 4G10 and streptavidin-PE ( B ) displayed large differences in the maximum fluorescence intensity. Within a single 96-well plate, well-specific standard curves displayed differences in slope and in the percentage of phosphorylation resulting in half maximal fluorescence (V 50 ), as calculated from the average of 100-300 measurements per standard per well. Serial dilutions of fluorescent anti-phosphotyrosine antibodies were used to confirm the effect of concentration on maximal fluorescence and slope. C , PY20-PE at the following concentrations in decreasing order: 150, 15, 1.5, 0.15 ng/mL. D , 4G10-biotin at the following concentrations in decreasing order: 1000, 100, 10, 1 ng/mL. Error bars for all mean fluorescence measurements indicate 99% confidence intervals.

    Article Snippet: Phosphorylated substrate was either labeled sequentially with a 1:1000 dilution of biotinylated 4G10 and a 1:1000 dilution of phycoerythrin-coupled streptavidin (both Millipore), or in a single step by the addition of a 1:10 dilution of phycoerythrin-conjugated PY20 (BD Phosflow, San Jose, CA) in TBST.

    Techniques: Construct, Fluorescence, Labeling, Concentration Assay

    Imatinib was incubated with K-562 cells or cell lysates. The sigmoidal decrease in Abltide phosphorylation as a result of intracellular kinase inhibition was monitored in cell lysates using PY20-PE or biotinylated 4G10 and streptavidin-PE. A , a three-fold difference between maximum mean fluorescence intensities in separate assays obscures direct comparisons. In blue, PY20-PE was used to label phosphorylated Abltide following the inhibition of lysates with imatinib (IC 50 = 0.52 μM). In red, biotinylated 4G10 and streptavidin-PE were used to label phosphorylated Abltide following incubation with lysates derived from K-562 cells treated with imatinib in culture (IC 50 = 2.48 μM). B, well-specific internal standard curves automatically correct for differences in raw fluorescence intensity between assays and are used to calculate the percentage of Abltide phosphorylation in each well. The blue curve represents the phosphorylation of Abltide by lysates treated with imatinib (IC 50 = 0.70 μM). The red curve represents the phosphorylation of Abltide by lysates derived from cells treated with imatinib in culture (IC 50 = 1.40 μM). Differences between IC 50 values in A and B are the result of data transformations by non-linear regression from well-specific internal standard curves. IC 50 values are not directly comparable between separate experiments because inhibition was performed under different conditions in each case. C , multidimensional analysis of Abltide phosphorylation reveals competitive dependence for ATP and imatinib in K-562 lysates. Serial dilutions of ATP and imatinib were organized along perpendicular axes of one half of a 96-well plate and incubated with K-562 lysates, which were prepared at high concentrations and diluted 50-fold to reduce the influence of endogenous ATP. Apparent IC 50 values increase with increasing concentrations of ATP, as shown by the heat map that highlights 1% increments of substrate phosphorylation from red (15-16% Abltide phosphorylation) to dark blue (0-1% Abltide phosphorylation).

    Journal: Molecular cancer therapeutics

    Article Title: A bead-based activity screen for small-molecule inhibitors of signal transduction in chronic myelogenous leukemia cells

    doi: 10.1158/1535-7163.MCT-10-0157

    Figure Lengend Snippet: Imatinib was incubated with K-562 cells or cell lysates. The sigmoidal decrease in Abltide phosphorylation as a result of intracellular kinase inhibition was monitored in cell lysates using PY20-PE or biotinylated 4G10 and streptavidin-PE. A , a three-fold difference between maximum mean fluorescence intensities in separate assays obscures direct comparisons. In blue, PY20-PE was used to label phosphorylated Abltide following the inhibition of lysates with imatinib (IC 50 = 0.52 μM). In red, biotinylated 4G10 and streptavidin-PE were used to label phosphorylated Abltide following incubation with lysates derived from K-562 cells treated with imatinib in culture (IC 50 = 2.48 μM). B, well-specific internal standard curves automatically correct for differences in raw fluorescence intensity between assays and are used to calculate the percentage of Abltide phosphorylation in each well. The blue curve represents the phosphorylation of Abltide by lysates treated with imatinib (IC 50 = 0.70 μM). The red curve represents the phosphorylation of Abltide by lysates derived from cells treated with imatinib in culture (IC 50 = 1.40 μM). Differences between IC 50 values in A and B are the result of data transformations by non-linear regression from well-specific internal standard curves. IC 50 values are not directly comparable between separate experiments because inhibition was performed under different conditions in each case. C , multidimensional analysis of Abltide phosphorylation reveals competitive dependence for ATP and imatinib in K-562 lysates. Serial dilutions of ATP and imatinib were organized along perpendicular axes of one half of a 96-well plate and incubated with K-562 lysates, which were prepared at high concentrations and diluted 50-fold to reduce the influence of endogenous ATP. Apparent IC 50 values increase with increasing concentrations of ATP, as shown by the heat map that highlights 1% increments of substrate phosphorylation from red (15-16% Abltide phosphorylation) to dark blue (0-1% Abltide phosphorylation).

    Article Snippet: Phosphorylated substrate was either labeled sequentially with a 1:1000 dilution of biotinylated 4G10 and a 1:1000 dilution of phycoerythrin-coupled streptavidin (both Millipore), or in a single step by the addition of a 1:10 dilution of phycoerythrin-conjugated PY20 (BD Phosflow, San Jose, CA) in TBST.

    Techniques: Incubation, Inhibition, Fluorescence, Derivative Assay