streptavidin  (New England Biolabs)


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    Streptavidin
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    Streptavidin 1 0 mg
    Catalog Number:
    n7021s
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    74
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    1 0 mg
    Category:
    Nucleic Acid Purification Reagents
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    New England Biolabs streptavidin
    Streptavidin
    Streptavidin 1 0 mg
    https://www.bioz.com/result/streptavidin/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    streptavidin - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction"

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction

    Journal: Biotechnology and applied biochemistry

    doi: 10.1002/bab.1645

    Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.
    Figure Legend Snippet: Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.

    Techniques Used: Construct, Concentration Assay

    Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.
    Figure Legend Snippet: Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.

    Techniques Used: Construct, Concentration Assay

    2) Product Images from "Proteins mediating DNA loops effectively block transcription"

    Article Title: Proteins mediating DNA loops effectively block transcription

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.3156

    LacI bound to an O1 operator pauses transcription. (A) A schematic representation of the DNA template used in magnetic tweezer transcription assays. The template contained a T7A1 promoter close to the upstream end, a stall site at position +22, an O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. A streptavidin‐labeled paramagnetic bead was coupled to the biotin label to for micromanipulation in the magnetic tweezer. Four examples of transcriptional elongation recorded using the magnetic tweezers are displayed. In ( B ) no LacI was included and transcription shortened the DNA tether progressively without interruption. When LacI was included ( C–E ) , transcription shortened the tether by about 0.2 um before pausing for about 200 s and then resuming. Transcription finally ceased after the tether shortened by either 0.35 um (C and D), a distance corresponding to the location of a terminator sequence, or 0.5 um (B and E), a distance corresponding to the end of the template.
    Figure Legend Snippet: LacI bound to an O1 operator pauses transcription. (A) A schematic representation of the DNA template used in magnetic tweezer transcription assays. The template contained a T7A1 promoter close to the upstream end, a stall site at position +22, an O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. A streptavidin‐labeled paramagnetic bead was coupled to the biotin label to for micromanipulation in the magnetic tweezer. Four examples of transcriptional elongation recorded using the magnetic tweezers are displayed. In ( B ) no LacI was included and transcription shortened the DNA tether progressively without interruption. When LacI was included ( C–E ) , transcription shortened the tether by about 0.2 um before pausing for about 200 s and then resuming. Transcription finally ceased after the tether shortened by either 0.35 um (C and D), a distance corresponding to the location of a terminator sequence, or 0.5 um (B and E), a distance corresponding to the end of the template.

    Techniques Used: Labeling, Micromanipulation, Sequencing

    Nanographs of RNA polymerases trapped by EDTA quenching during elongation along DNA with and without LacI‐mediated loops. (A) Schematic representation of the DNA templates used in scanning force microscopy assays. All templates contained a T7A1 promoter close to the upstream end, a stall site at position +22, a “far” O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. The two DNA templates used for SFM measurements of transcription differed in the “near” operator positioned 253 bp downstream from the promoter; one template contained the Os operator while the other contained the O2 operator. The terminator was the very last feature of the sequence and was biotin labeled. Streptavidin was coupled to the biotin label to facilitate identifying the “downstream” end of the molecule in SFM nanographs. (B) The upper row is a selection of molecules along which RNA polymerases (large yellow particle) had not progressed very far from the transcription start site near the end of the DNA without a streptavidin particle (blue). Closed and open conformations of the LacI tetramers are visible for either looped (left) or unlooped (right) columns. The closed conformations are shown as blue particles that are slightly larger than the streptavidin. In the open conformation, two lobes are visible especially on looped DNA. These lobes correspond to individual dimers with DNA binding head groups. The TECs shown in the lower row had progressed further and small coils of RNA emanate from them (see inset schematics for the regions of interest). These TECs have collided with LacI particles. The LacI particles correspond to blue protuberances on the periphery of the larger yellow RNA polymerase particle. The RNA polymerases themselves appear to shift to the side opposite LacI especially for open LacI conformations.
    Figure Legend Snippet: Nanographs of RNA polymerases trapped by EDTA quenching during elongation along DNA with and without LacI‐mediated loops. (A) Schematic representation of the DNA templates used in scanning force microscopy assays. All templates contained a T7A1 promoter close to the upstream end, a stall site at position +22, a “far” O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. The two DNA templates used for SFM measurements of transcription differed in the “near” operator positioned 253 bp downstream from the promoter; one template contained the Os operator while the other contained the O2 operator. The terminator was the very last feature of the sequence and was biotin labeled. Streptavidin was coupled to the biotin label to facilitate identifying the “downstream” end of the molecule in SFM nanographs. (B) The upper row is a selection of molecules along which RNA polymerases (large yellow particle) had not progressed very far from the transcription start site near the end of the DNA without a streptavidin particle (blue). Closed and open conformations of the LacI tetramers are visible for either looped (left) or unlooped (right) columns. The closed conformations are shown as blue particles that are slightly larger than the streptavidin. In the open conformation, two lobes are visible especially on looped DNA. These lobes correspond to individual dimers with DNA binding head groups. The TECs shown in the lower row had progressed further and small coils of RNA emanate from them (see inset schematics for the regions of interest). These TECs have collided with LacI particles. The LacI particles correspond to blue protuberances on the periphery of the larger yellow RNA polymerase particle. The RNA polymerases themselves appear to shift to the side opposite LacI especially for open LacI conformations.

    Techniques Used: Microscopy, Sequencing, Labeling, Selection, Binding Assay

    Nanographs of RNAP elongation along DNA with and without LacI‐mediated loops. (Top) Schematic representations of transcription elongation complexes (TECs). Columns correspond to different DNA topologies and LacI occupancy. The first column corresponds to transcription elongation without LacI in the reaction buffer. The second and third column correspond to DNA found in a looped topology, with LacI at each of the two operators; the fourth and fifth columns correspond to unlooped DNA with both operators occupied. Transcription elongation progress is categorized in five zones (roman numerals I‐V). Numerals in the schematic correspond to each row of the nanograph array. Each image in the array is representative of its corresponding category (columns) and elongation progress (rows). Image colors indicate height, according to the color scale below. RNAP, LacI and streptavidin particles are indicated by yellow, light blue, and white arrows respectively. (Row I) AFM images of RNAP bound at the T7A1 promoter. (Row II) Images in which TECs have not yet reached the near operator. (Row III) Images in which TECs contact LacI at the near operator. (Row IV) Images in which TECs were found between the two operators. (Row V) Images in which TECs were beyond the far operator. As indicated in the figure, images for RNAP in zone V were not detected for looped O2‐O1 DNA and unlooped Os‐O1 DNA. Note that nascent RNA associated with each TEC is visible, especially in the first column (insets), and increases in size as the RNAP progresses (I to V).
    Figure Legend Snippet: Nanographs of RNAP elongation along DNA with and without LacI‐mediated loops. (Top) Schematic representations of transcription elongation complexes (TECs). Columns correspond to different DNA topologies and LacI occupancy. The first column corresponds to transcription elongation without LacI in the reaction buffer. The second and third column correspond to DNA found in a looped topology, with LacI at each of the two operators; the fourth and fifth columns correspond to unlooped DNA with both operators occupied. Transcription elongation progress is categorized in five zones (roman numerals I‐V). Numerals in the schematic correspond to each row of the nanograph array. Each image in the array is representative of its corresponding category (columns) and elongation progress (rows). Image colors indicate height, according to the color scale below. RNAP, LacI and streptavidin particles are indicated by yellow, light blue, and white arrows respectively. (Row I) AFM images of RNAP bound at the T7A1 promoter. (Row II) Images in which TECs have not yet reached the near operator. (Row III) Images in which TECs contact LacI at the near operator. (Row IV) Images in which TECs were found between the two operators. (Row V) Images in which TECs were beyond the far operator. As indicated in the figure, images for RNAP in zone V were not detected for looped O2‐O1 DNA and unlooped Os‐O1 DNA. Note that nascent RNA associated with each TEC is visible, especially in the first column (insets), and increases in size as the RNAP progresses (I to V).

    Techniques Used:

    3) Product Images from "A DNA nanoscope via auto-cycling proximity recording"

    Article Title: A DNA nanoscope via auto-cycling proximity recording

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00542-3

    Proof-of-principle experiments. a Generation of Full-records requires the colocalization of probes, here by biotinylated hairpin loops bound to streptavidin, whereas isolated probes can always generate Half-records. Cropped denaturing PAGE gel depicting 10 μl reactions (40 min at 37 °C) with biotin–streptavidin association and 4:1 overall probe/streptavidin stoichiometry ( inset ), 8 and 22 nt barcodes (19 and 33 nt stem lengths copied), 10:1 initial primer/probe, and 40 nM total probe concentration. A single primer sequence was used and no secondary amplification was performed. b Auto-cycling is demonstrated by quantification of Cy5-labeled probes on cropped denaturing PAGE gels. Rapidly cycling probes with Iso-dC/dG stoppers and phosphorothioate primers were used, with probes at 0.1 nM and primers at 1000-fold excess to probes in each time series. Quantification of Full-records yields the plot. Half-records are difficult to detect because of low probe concentration. See probe details and sequences for a , b in Supplementary Fig. 2 and full gels in Supplementary Fig. 3 . c An example of a single probe (with Barcode i ) making multiple partnerships (with Barcodes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${j^*}$$\end{document} j * ), read with Illumina MiSeq next-generation sequencing. Here, probes encoded a universal primer sequence and unique barcodes (in place of spacer s of Fig. 2a ), and were held in tetramers by streptavidin. Primer sequences cropped for clarity. See Supplementary Fig. 4 for the unique-barcode APR cycle, probe details, and sequencing methods
    Figure Legend Snippet: Proof-of-principle experiments. a Generation of Full-records requires the colocalization of probes, here by biotinylated hairpin loops bound to streptavidin, whereas isolated probes can always generate Half-records. Cropped denaturing PAGE gel depicting 10 μl reactions (40 min at 37 °C) with biotin–streptavidin association and 4:1 overall probe/streptavidin stoichiometry ( inset ), 8 and 22 nt barcodes (19 and 33 nt stem lengths copied), 10:1 initial primer/probe, and 40 nM total probe concentration. A single primer sequence was used and no secondary amplification was performed. b Auto-cycling is demonstrated by quantification of Cy5-labeled probes on cropped denaturing PAGE gels. Rapidly cycling probes with Iso-dC/dG stoppers and phosphorothioate primers were used, with probes at 0.1 nM and primers at 1000-fold excess to probes in each time series. Quantification of Full-records yields the plot. Half-records are difficult to detect because of low probe concentration. See probe details and sequences for a , b in Supplementary Fig. 2 and full gels in Supplementary Fig. 3 . c An example of a single probe (with Barcode i ) making multiple partnerships (with Barcodes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${j^*}$$\end{document} j * ), read with Illumina MiSeq next-generation sequencing. Here, probes encoded a universal primer sequence and unique barcodes (in place of spacer s of Fig. 2a ), and were held in tetramers by streptavidin. Primer sequences cropped for clarity. See Supplementary Fig. 4 for the unique-barcode APR cycle, probe details, and sequencing methods

    Techniques Used: Isolation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Sequencing, Amplification, Labeling, Next-Generation Sequencing

    4) Product Images from "Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures"

    Article Title: Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures

    Journal:

    doi: 10.1016/j.nano.2006.10.003

    AFM images of flat silicon surface with native oxide before (a) and after (b) surface modification with BAC-BSA and binding to streptavidin and fluorescence imaging confirmation of streptavidin binding to a BAC-BSA-modified native oxide layer (d) but
    Figure Legend Snippet: AFM images of flat silicon surface with native oxide before (a) and after (b) surface modification with BAC-BSA and binding to streptavidin and fluorescence imaging confirmation of streptavidin binding to a BAC-BSA-modified native oxide layer (d) but

    Techniques Used: Modification, BAC Assay, Binding Assay, Fluorescence, Imaging

    measured I–V curves for the sensor before modification, after the attachment of BSA, upon exposure to d-Biotin Streptavidin, and after exposure to Streptavidin. Notice there is a 4.3% average decrease in conductivity after exposure to Streptavidin
    Figure Legend Snippet: measured I–V curves for the sensor before modification, after the attachment of BSA, upon exposure to d-Biotin Streptavidin, and after exposure to Streptavidin. Notice there is a 4.3% average decrease in conductivity after exposure to Streptavidin

    Techniques Used: Modification

    measured transient response of the a-Si sensor to the introduction of streptavidin at t = 360 seconds. The current signal settled to its final value 6 seconds after streptavidin was introduced.
    Figure Legend Snippet: measured transient response of the a-Si sensor to the introduction of streptavidin at t = 360 seconds. The current signal settled to its final value 6 seconds after streptavidin was introduced.

    Techniques Used:

    5) Product Images from "Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice"

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice

    Journal: Nature Communications

    doi: 10.1038/ncomms10618

    Binding affinities of the indicated germline VRC01-class antibodies to selected 426c variants. Soluble trimeric 426c gp140 variants were biotinylated and immobilized on a streptavidin biosensor. The association constant of the various germline VRC01-class antibodies was determined by BLI, as described in the Methods section. Undetectable antibody-Env binding is shown on the x axis. Full kinetic parameters are shown in Supplementary Table 1 . See Table 1 for a description of the various mutations on the 426c Env.
    Figure Legend Snippet: Binding affinities of the indicated germline VRC01-class antibodies to selected 426c variants. Soluble trimeric 426c gp140 variants were biotinylated and immobilized on a streptavidin biosensor. The association constant of the various germline VRC01-class antibodies was determined by BLI, as described in the Methods section. Undetectable antibody-Env binding is shown on the x axis. Full kinetic parameters are shown in Supplementary Table 1 . See Table 1 for a description of the various mutations on the 426c Env.

    Techniques Used: Binding Assay

    6) Product Images from "Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *"

    Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.730135

    NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′
    Figure Legend Snippet: NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′

    Techniques Used:

    7) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001164

    Mlh1-Mlh3’s endonuclease activity requires a continuous substrate and increases as substrate size increases. (A) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on circular pUC18 (linearized prior to gel loading) (2.7 kb; black), Hin dIII linearized pUC18 (red), and Hin dIII linearized pUC18 with streptavidin (SA) bound to ends (blue). Migration of linearized substrate (l) is indicated. (B) Average of two separate experiments: fraction nicked defined as fraction of substrate lost plotted against yeast Mlh1-Mlh3 concentration; error bars represent the standard deviation between three experiments. (C) Top: native agarose gel electrophoresis analysis of yeast Mlh1-Mlh3 (150 nM) endonuclease activity on circular substrate ranging from 2.7 kb to 12 kb. The concentration of nucleotide in each reaction is 15 μM. (D) Quantification of nicking in lanes 4, 7, 10, and 13 in C averaged from three separate experiments. Error bars indicate standard deviation. (E) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on 12-kb circular DNA (black) and Hin dIII linearized 12 kb substrate (red). (F) Average of three separate experiments; error bars represent standard deviation. All nicking reactions were carried out for 60 min.
    Figure Legend Snippet: Mlh1-Mlh3’s endonuclease activity requires a continuous substrate and increases as substrate size increases. (A) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on circular pUC18 (linearized prior to gel loading) (2.7 kb; black), Hin dIII linearized pUC18 (red), and Hin dIII linearized pUC18 with streptavidin (SA) bound to ends (blue). Migration of linearized substrate (l) is indicated. (B) Average of two separate experiments: fraction nicked defined as fraction of substrate lost plotted against yeast Mlh1-Mlh3 concentration; error bars represent the standard deviation between three experiments. (C) Top: native agarose gel electrophoresis analysis of yeast Mlh1-Mlh3 (150 nM) endonuclease activity on circular substrate ranging from 2.7 kb to 12 kb. The concentration of nucleotide in each reaction is 15 μM. (D) Quantification of nicking in lanes 4, 7, 10, and 13 in C averaged from three separate experiments. Error bars indicate standard deviation. (E) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on 12-kb circular DNA (black) and Hin dIII linearized 12 kb substrate (red). (F) Average of three separate experiments; error bars represent standard deviation. All nicking reactions were carried out for 60 min.

    Techniques Used: Activity Assay, Migration, Concentration Assay, Standard Deviation, Agarose Gel Electrophoresis

    Mlh1-Mlh3’s endonuclease activity is inhibited by a loop mismatch and biotin-streptavidin linkages in plasmid DNA. (A) Mlh1-Mlh3 nicking activity on homoduplex (black), biotinylated (green), or biotin-streptavidin–containing (blue) 7.2 kb circular substrates (15 μM total nucleotide). Lanes 4–7, 10–13, and 16–19 contain 50, 150, 300, and 500 nM Mlh1-Mlh3, respectively. The amount of nicked product (n) and linear product (black triangle) was quantified as a fraction of the total starting closed circular substrate (cc). (B) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (C) Mlh1-Mlh3 nicking activity on homoduplex (black) or +8 loop mismatch–containing (red) 7 kb circular substrate (15 μM total nucleotide). Lanes 3–7 and 9–13 contain 25, 50, 150, 200, and 300 nM Mlh1-Mlh3, respectively. (D) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (E) Mlh1-Mlh3 nicking activity on 7 kb linear substrates containing a +8 loop mismatch 550 ( Drd I), 3,900 ( Afe I), or 6,600 ( Bgl II) base pairs from one end. Average of two experiments is indicated below the gel. See Materials and methods for details. All nicking reactions were carried out for 60 min.
    Figure Legend Snippet: Mlh1-Mlh3’s endonuclease activity is inhibited by a loop mismatch and biotin-streptavidin linkages in plasmid DNA. (A) Mlh1-Mlh3 nicking activity on homoduplex (black), biotinylated (green), or biotin-streptavidin–containing (blue) 7.2 kb circular substrates (15 μM total nucleotide). Lanes 4–7, 10–13, and 16–19 contain 50, 150, 300, and 500 nM Mlh1-Mlh3, respectively. The amount of nicked product (n) and linear product (black triangle) was quantified as a fraction of the total starting closed circular substrate (cc). (B) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (C) Mlh1-Mlh3 nicking activity on homoduplex (black) or +8 loop mismatch–containing (red) 7 kb circular substrate (15 μM total nucleotide). Lanes 3–7 and 9–13 contain 25, 50, 150, 200, and 300 nM Mlh1-Mlh3, respectively. (D) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (E) Mlh1-Mlh3 nicking activity on 7 kb linear substrates containing a +8 loop mismatch 550 ( Drd I), 3,900 ( Afe I), or 6,600 ( Bgl II) base pairs from one end. Average of two experiments is indicated below the gel. See Materials and methods for details. All nicking reactions were carried out for 60 min.

    Techniques Used: Activity Assay, Plasmid Preparation, Standard Deviation

    8) Product Images from "Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice"

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice

    Journal: Nature Communications

    doi: 10.1038/ncomms10618

    Binding affinities of the indicated germline VRC01-class antibodies to selected 426c variants. Soluble trimeric 426c gp140 variants were biotinylated and immobilized on a streptavidin biosensor. The association constant of the various germline VRC01-class antibodies was determined by BLI, as described in the Methods section. Undetectable antibody-Env binding is shown on the x axis. Full kinetic parameters are shown in Supplementary Table 1 . See Table 1 for a description of the various mutations on the 426c Env.
    Figure Legend Snippet: Binding affinities of the indicated germline VRC01-class antibodies to selected 426c variants. Soluble trimeric 426c gp140 variants were biotinylated and immobilized on a streptavidin biosensor. The association constant of the various germline VRC01-class antibodies was determined by BLI, as described in the Methods section. Undetectable antibody-Env binding is shown on the x axis. Full kinetic parameters are shown in Supplementary Table 1 . See Table 1 for a description of the various mutations on the 426c Env.

    Techniques Used: Binding Assay

    9) Product Images from "A DNA nanoscope via auto-cycling proximity recording"

    Article Title: A DNA nanoscope via auto-cycling proximity recording

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00542-3

    Proof-of-principle experiments. a Generation of Full-records requires the colocalization of probes, here by biotinylated hairpin loops bound to streptavidin, whereas isolated probes can always generate Half-records. Cropped denaturing PAGE gel depicting 10 μl reactions (40 min at 37 °C) with biotin–streptavidin association and 4:1 overall probe/streptavidin stoichiometry ( inset ), 8 and 22 nt barcodes (19 and 33 nt stem lengths copied), 10:1 initial primer/probe, and 40 nM total probe concentration. A single primer sequence was used and no secondary amplification was performed. b Auto-cycling is demonstrated by quantification of Cy5-labeled probes on cropped denaturing PAGE gels. Rapidly cycling probes with Iso-dC/dG stoppers and phosphorothioate primers were used, with probes at 0.1 nM and primers at 1000-fold excess to probes in each time series. Quantification of Full-records yields the plot. Half-records are difficult to detect because of low probe concentration. See probe details and sequences for a , b in Supplementary Fig. 2 and full gels in Supplementary Fig. 3 . c An example of a single probe (with Barcode i ) making multiple partnerships (with Barcodes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${j^*}$$\end{document} j * ), read with Illumina MiSeq next-generation sequencing. Here, probes encoded a universal primer sequence and unique barcodes (in place of spacer s of Fig. 2a ), and were held in tetramers by streptavidin. Primer sequences cropped for clarity. See Supplementary Fig. 4 for the unique-barcode APR cycle, probe details, and sequencing methods
    Figure Legend Snippet: Proof-of-principle experiments. a Generation of Full-records requires the colocalization of probes, here by biotinylated hairpin loops bound to streptavidin, whereas isolated probes can always generate Half-records. Cropped denaturing PAGE gel depicting 10 μl reactions (40 min at 37 °C) with biotin–streptavidin association and 4:1 overall probe/streptavidin stoichiometry ( inset ), 8 and 22 nt barcodes (19 and 33 nt stem lengths copied), 10:1 initial primer/probe, and 40 nM total probe concentration. A single primer sequence was used and no secondary amplification was performed. b Auto-cycling is demonstrated by quantification of Cy5-labeled probes on cropped denaturing PAGE gels. Rapidly cycling probes with Iso-dC/dG stoppers and phosphorothioate primers were used, with probes at 0.1 nM and primers at 1000-fold excess to probes in each time series. Quantification of Full-records yields the plot. Half-records are difficult to detect because of low probe concentration. See probe details and sequences for a , b in Supplementary Fig. 2 and full gels in Supplementary Fig. 3 . c An example of a single probe (with Barcode i ) making multiple partnerships (with Barcodes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${j^*}$$\end{document} j * ), read with Illumina MiSeq next-generation sequencing. Here, probes encoded a universal primer sequence and unique barcodes (in place of spacer s of Fig. 2a ), and were held in tetramers by streptavidin. Primer sequences cropped for clarity. See Supplementary Fig. 4 for the unique-barcode APR cycle, probe details, and sequencing methods

    Techniques Used: Isolation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Sequencing, Amplification, Labeling, Next-Generation Sequencing

    10) Product Images from "The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity *"

    Article Title: The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.435966

    PriA and holoenzyme do not coexist on PriA-inhibited replication forks. A , diagram depicting two possible models of PriA inhibiting the strand displacement reaction and the expected result of each for reactions on streptavidin ( SA ) beads. The primer is labeled on the 5′-end with 32 P so that primer extension and helicase activity can be monitored. The primer contains a biotin near the 5′-end so that substrates can be conjugated to streptavidin-linked beads. Scheme 1 depicts PriA blocking the 3′-OH of the primer, physically preventing Pol III HE from binding. Scheme 2 portrays an inhibition model where both PriA and Pol III HE bind to the substrate. Primosomal proteins (PriB, DnaT, DnaB, and DnaC) were added as described under “Experimental Procedures.” B , denaturing gel analysis to monitor primer extension by E. coli Pol III (exo-). Lanes 11–13 are dilutions of the positive control lane 3 to establish detection limits. For both B and C , lanes 8 and 9 contain the full Pol III HE but in lane 8 , the β-subunit was omitted, and in lane 9 , SSB was omitted. C , native gel analysis to monitor substrate unwinding by E. coli DnaB helicase. The upper band is the replication fork, and the 90/90 duplex product in those cases is where replication occurs. The lower band is the displaced leading strand primer template. In lane 5 , ∼45% of the substrate was unwound by the helicase. In lane 7 , ∼40% of the substrate was unwound by the helicase. In all other lanes, the amount of substrate unwound is not significantly above background. D , denaturing gel analysis to monitor primer extension by E. coli Pol III (exo-) on immobilized substrate without a washing step. Experiment carried out as described under “Experimental Procedures,” except after incubation with Pol III HE components, the washing steps were omitted. In lane 5 , ∼45% of the primer is elongated.
    Figure Legend Snippet: PriA and holoenzyme do not coexist on PriA-inhibited replication forks. A , diagram depicting two possible models of PriA inhibiting the strand displacement reaction and the expected result of each for reactions on streptavidin ( SA ) beads. The primer is labeled on the 5′-end with 32 P so that primer extension and helicase activity can be monitored. The primer contains a biotin near the 5′-end so that substrates can be conjugated to streptavidin-linked beads. Scheme 1 depicts PriA blocking the 3′-OH of the primer, physically preventing Pol III HE from binding. Scheme 2 portrays an inhibition model where both PriA and Pol III HE bind to the substrate. Primosomal proteins (PriB, DnaT, DnaB, and DnaC) were added as described under “Experimental Procedures.” B , denaturing gel analysis to monitor primer extension by E. coli Pol III (exo-). Lanes 11–13 are dilutions of the positive control lane 3 to establish detection limits. For both B and C , lanes 8 and 9 contain the full Pol III HE but in lane 8 , the β-subunit was omitted, and in lane 9 , SSB was omitted. C , native gel analysis to monitor substrate unwinding by E. coli DnaB helicase. The upper band is the replication fork, and the 90/90 duplex product in those cases is where replication occurs. The lower band is the displaced leading strand primer template. In lane 5 , ∼45% of the substrate was unwound by the helicase. In lane 7 , ∼40% of the substrate was unwound by the helicase. In all other lanes, the amount of substrate unwound is not significantly above background. D , denaturing gel analysis to monitor primer extension by E. coli Pol III (exo-) on immobilized substrate without a washing step. Experiment carried out as described under “Experimental Procedures,” except after incubation with Pol III HE components, the washing steps were omitted. In lane 5 , ∼45% of the primer is elongated.

    Techniques Used: Labeling, Activity Assay, Blocking Assay, Binding Assay, Inhibition, Positive Control, Incubation

    11) Product Images from "A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning"

    Article Title: A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning

    Journal: Journal of immunological methods

    doi: 10.1016/j.jim.2017.05.010

    Workflow summary of mAb discovery pipeline in mice. Upon completion of the immunization regimen, spleens are harvested and gently homogenized to a single-cell suspension. The cell suspension is treated by negative magnetically-assisted selection until predominantly B cells remain. The B-cell suspension is stained using a cocktail of antibodies and Ag-streptavidin (as well as decoy-streptavidin) complexes to facilitate the sorting of Ag-positive class-switched cells. The cells are then cultured and induced to express antibodies in presence of supportive cytokines and feeder cells. Culture-conditioned supernatants containing target antibodies are then screened using Ag-specific ELISA, and positive wells are harvested for RNA isolation. Ab-encoding RNA is then reverse-transcribed, amplified and cloned into an expression cassette enabling recombinant production of the mAbs, followed by functional validation.
    Figure Legend Snippet: Workflow summary of mAb discovery pipeline in mice. Upon completion of the immunization regimen, spleens are harvested and gently homogenized to a single-cell suspension. The cell suspension is treated by negative magnetically-assisted selection until predominantly B cells remain. The B-cell suspension is stained using a cocktail of antibodies and Ag-streptavidin (as well as decoy-streptavidin) complexes to facilitate the sorting of Ag-positive class-switched cells. The cells are then cultured and induced to express antibodies in presence of supportive cytokines and feeder cells. Culture-conditioned supernatants containing target antibodies are then screened using Ag-specific ELISA, and positive wells are harvested for RNA isolation. Ab-encoding RNA is then reverse-transcribed, amplified and cloned into an expression cassette enabling recombinant production of the mAbs, followed by functional validation.

    Techniques Used: Mouse Assay, Selection, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Amplification, Clone Assay, Expressing, Recombinant, Functional Assay

    12) Product Images from "IR-783 Labeling of a Peptide Receptor for ‘Turn-On’ Fluorescence Based Sensing"

    Article Title: IR-783 Labeling of a Peptide Receptor for ‘Turn-On’ Fluorescence Based Sensing

    Journal: Chemosensors (Basel, Switzerland)

    doi: 10.3390/chemosensors6040047

    Schematic of IR-783 labeled peptide sequence VSHPQAPF serving as biotin mimic for recognition by streptavidin. Binding results in induction of turn-on fluorescence enhancement that is reversible upon addition of native biotin to displace the probe returning it to weak fluorescence.
    Figure Legend Snippet: Schematic of IR-783 labeled peptide sequence VSHPQAPF serving as biotin mimic for recognition by streptavidin. Binding results in induction of turn-on fluorescence enhancement that is reversible upon addition of native biotin to displace the probe returning it to weak fluorescence.

    Techniques Used: Labeling, Sequencing, Binding Assay, Fluorescence

    The fluorescence intensity of the IR783-VSHPQAPF was observed to increase linearly with increasing concentrations of streptavidin.
    Figure Legend Snippet: The fluorescence intensity of the IR783-VSHPQAPF was observed to increase linearly with increasing concentrations of streptavidin.

    Techniques Used: Fluorescence

    The biotin mimetic probe IR-783 VSHPQAPF, when bound to streptavidin, has enhanced fluorescence, which is reversible when native biotin is subsequently added to competitively elute the probe from the streptavidin target: ( a ) Addition of increasing amount of biotin shows a significant decrease in fluorescence when more than one equivalent of biotin was combined with respect to the streptavidin concentration; ( b ) Fluorescence spectra show that the addition of 3 equivalents of biotin results in a decrease in intensity of the signal but no significant peak shift.
    Figure Legend Snippet: The biotin mimetic probe IR-783 VSHPQAPF, when bound to streptavidin, has enhanced fluorescence, which is reversible when native biotin is subsequently added to competitively elute the probe from the streptavidin target: ( a ) Addition of increasing amount of biotin shows a significant decrease in fluorescence when more than one equivalent of biotin was combined with respect to the streptavidin concentration; ( b ) Fluorescence spectra show that the addition of 3 equivalents of biotin results in a decrease in intensity of the signal but no significant peak shift.

    Techniques Used: Fluorescence, Concentration Assay

    Microscale thermophoresis signals resulting from binding induced changes in hydration shell corresponding to distinct thermophoretic signals for the probe (IR783-VSHPQAPF) with increasing concentrations of streptavidin target.
    Figure Legend Snippet: Microscale thermophoresis signals resulting from binding induced changes in hydration shell corresponding to distinct thermophoretic signals for the probe (IR783-VSHPQAPF) with increasing concentrations of streptavidin target.

    Techniques Used: Microscale Thermophoresis, Binding Assay

    Microscale thermophoresis showing distinct signals for the IR783-VSHPQAPF probe before and after binding to streptavidin revealing a clear binding event and again returning to its original signal upon addition of native biotin confirming the release of the probe from streptavidin.
    Figure Legend Snippet: Microscale thermophoresis showing distinct signals for the IR783-VSHPQAPF probe before and after binding to streptavidin revealing a clear binding event and again returning to its original signal upon addition of native biotin confirming the release of the probe from streptavidin.

    Techniques Used: Microscale Thermophoresis, Binding Assay

    13) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001164

    Mlh1-Mlh3’s endonuclease activity requires a continuous substrate and increases as substrate size increases. (A) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on circular pUC18 (linearized prior to gel loading) (2.7 kb; black), Hin dIII linearized pUC18 (red), and Hin dIII linearized pUC18 with streptavidin (SA) bound to ends (blue). Migration of linearized substrate (l) is indicated. (B) Average of two separate experiments: fraction nicked defined as fraction of substrate lost plotted against yeast Mlh1-Mlh3 concentration; error bars represent the standard deviation between three experiments. (C) Top: native agarose gel electrophoresis analysis of yeast Mlh1-Mlh3 (150 nM) endonuclease activity on circular substrate ranging from 2.7 kb to 12 kb. The concentration of nucleotide in each reaction is 15 μM. (D) Quantification of nicking in lanes 4, 7, 10, and 13 in C averaged from three separate experiments. Error bars indicate standard deviation. (E) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on 12-kb circular DNA (black) and Hin dIII linearized 12 kb substrate (red). (F) Average of three separate experiments; error bars represent standard deviation. All nicking reactions were carried out for 60 min.
    Figure Legend Snippet: Mlh1-Mlh3’s endonuclease activity requires a continuous substrate and increases as substrate size increases. (A) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on circular pUC18 (linearized prior to gel loading) (2.7 kb; black), Hin dIII linearized pUC18 (red), and Hin dIII linearized pUC18 with streptavidin (SA) bound to ends (blue). Migration of linearized substrate (l) is indicated. (B) Average of two separate experiments: fraction nicked defined as fraction of substrate lost plotted against yeast Mlh1-Mlh3 concentration; error bars represent the standard deviation between three experiments. (C) Top: native agarose gel electrophoresis analysis of yeast Mlh1-Mlh3 (150 nM) endonuclease activity on circular substrate ranging from 2.7 kb to 12 kb. The concentration of nucleotide in each reaction is 15 μM. (D) Quantification of nicking in lanes 4, 7, 10, and 13 in C averaged from three separate experiments. Error bars indicate standard deviation. (E) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on 12-kb circular DNA (black) and Hin dIII linearized 12 kb substrate (red). (F) Average of three separate experiments; error bars represent standard deviation. All nicking reactions were carried out for 60 min.

    Techniques Used: Activity Assay, Migration, Concentration Assay, Standard Deviation, Agarose Gel Electrophoresis

    Mlh1-Mlh3’s endonuclease activity is inhibited by a loop mismatch and biotin-streptavidin linkages in plasmid DNA. (A) Mlh1-Mlh3 nicking activity on homoduplex (black), biotinylated (green), or biotin-streptavidin–containing (blue) 7.2 kb circular substrates (15 μM total nucleotide). Lanes 4–7, 10–13, and 16–19 contain 50, 150, 300, and 500 nM Mlh1-Mlh3, respectively. The amount of nicked product (n) and linear product (black triangle) was quantified as a fraction of the total starting closed circular substrate (cc). (B) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (C) Mlh1-Mlh3 nicking activity on homoduplex (black) or +8 loop mismatch–containing (red) 7 kb circular substrate (15 μM total nucleotide). Lanes 3–7 and 9–13 contain 25, 50, 150, 200, and 300 nM Mlh1-Mlh3, respectively. (D) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (E) Mlh1-Mlh3 nicking activity on 7 kb linear substrates containing a +8 loop mismatch 550 ( Drd I), 3,900 ( Afe I), or 6,600 ( Bgl II) base pairs from one end. Average of two experiments is indicated below the gel. See Materials and methods for details. All nicking reactions were carried out for 60 min.
    Figure Legend Snippet: Mlh1-Mlh3’s endonuclease activity is inhibited by a loop mismatch and biotin-streptavidin linkages in plasmid DNA. (A) Mlh1-Mlh3 nicking activity on homoduplex (black), biotinylated (green), or biotin-streptavidin–containing (blue) 7.2 kb circular substrates (15 μM total nucleotide). Lanes 4–7, 10–13, and 16–19 contain 50, 150, 300, and 500 nM Mlh1-Mlh3, respectively. The amount of nicked product (n) and linear product (black triangle) was quantified as a fraction of the total starting closed circular substrate (cc). (B) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (C) Mlh1-Mlh3 nicking activity on homoduplex (black) or +8 loop mismatch–containing (red) 7 kb circular substrate (15 μM total nucleotide). Lanes 3–7 and 9–13 contain 25, 50, 150, 200, and 300 nM Mlh1-Mlh3, respectively. (D) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (E) Mlh1-Mlh3 nicking activity on 7 kb linear substrates containing a +8 loop mismatch 550 ( Drd I), 3,900 ( Afe I), or 6,600 ( Bgl II) base pairs from one end. Average of two experiments is indicated below the gel. See Materials and methods for details. All nicking reactions were carried out for 60 min.

    Techniques Used: Activity Assay, Plasmid Preparation, Standard Deviation

    14) Product Images from "Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling"

    Article Title: Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1655

    A catalytic RNA from the A. pernix genome that reacts with a disubstituted epoxide ( a ) PAGE streptavidin gel mobility shift following incubation of random sequence RNA (“random”), the A. pernix species from Round 6 of the selection (“selected”), and the A. pernix fragment corresponding to the reference genome sequence (“genomic”) with epoxide probe 1 (1 mM) for 16 h at room temperature (1 µM RNA). The complete gel is shown in Supplementary Figure 12 . ( b ) Secondary structure model of the minimized A. pernix catalytic RNA. The reactive guanosine (blue) was identified by RNAse T1 digestion and mass spectrometry. ( c ) Sequence logo based on high-throughput DNA sequencing of the RNA species surviving selection of a partially randomized RNA pool derived from the minimized 42-nt A. pernix catalytic RNA.
    Figure Legend Snippet: A catalytic RNA from the A. pernix genome that reacts with a disubstituted epoxide ( a ) PAGE streptavidin gel mobility shift following incubation of random sequence RNA (“random”), the A. pernix species from Round 6 of the selection (“selected”), and the A. pernix fragment corresponding to the reference genome sequence (“genomic”) with epoxide probe 1 (1 mM) for 16 h at room temperature (1 µM RNA). The complete gel is shown in Supplementary Figure 12 . ( b ) Secondary structure model of the minimized A. pernix catalytic RNA. The reactive guanosine (blue) was identified by RNAse T1 digestion and mass spectrometry. ( c ) Sequence logo based on high-throughput DNA sequencing of the RNA species surviving selection of a partially randomized RNA pool derived from the minimized 42-nt A. pernix catalytic RNA.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Mobility Shift, Incubation, Sequencing, Selection, Mass Spectrometry, High Throughput Screening Assay, DNA Sequencing, Derivative Assay

    Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins ( a ) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. ( b ) Total RNA from HEK 293T cells was reacted with epoxide-azide 14 , followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14 . ( c ) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1 -modified ASH1 -catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15 .
    Figure Legend Snippet: Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins ( a ) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. ( b ) Total RNA from HEK 293T cells was reacted with epoxide-azide 14 , followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14 . ( c ) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1 -modified ASH1 -catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15 .

    Techniques Used: RNA Binding Assay, Labeling, Modification, Polyacrylamide Gel Electrophoresis, Fluorescence, Imaging, In Vitro, Mutagenesis, Western Blot, Binding Assay, Incubation, Magnetic Beads

    15) Product Images from "Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins"

    Article Title: Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

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

    doi: 10.1073/pnas.1516674113

    Human Exo1-biotin (hExo1-bio) purification and labeling. ( A ) Purification scheme for hExo1-bio. ( B ) SDS/PAGE gel showing hExo1-bio and hExo1-bio + streptavidin. Gel shift of hExo1-bio-streptavidin conjugates are indicated. The complete disappearance of the hExo1-bio band indicates that nearly 100% of the purified nucleases are biotinylated. ( C ). Together, these assays indicate that streptavidin-conjugated hExo1-bio retains full resection activity. ( D ) Snapshots at indicated times ( Upper ) and single-particle tracking of two representative trajectories of resection by CF488-anti-biotin–labeled hExo1 ( Lower ). In both trajectories, hExo1 transitions between a resecting and a paused state. These results indicate that both states are intrinsic to hExo1 and are not dependent on the nature of the fluorophore.
    Figure Legend Snippet: Human Exo1-biotin (hExo1-bio) purification and labeling. ( A ) Purification scheme for hExo1-bio. ( B ) SDS/PAGE gel showing hExo1-bio and hExo1-bio + streptavidin. Gel shift of hExo1-bio-streptavidin conjugates are indicated. The complete disappearance of the hExo1-bio band indicates that nearly 100% of the purified nucleases are biotinylated. ( C ). Together, these assays indicate that streptavidin-conjugated hExo1-bio retains full resection activity. ( D ) Snapshots at indicated times ( Upper ) and single-particle tracking of two representative trajectories of resection by CF488-anti-biotin–labeled hExo1 ( Lower ). In both trajectories, hExo1 transitions between a resecting and a paused state. These results indicate that both states are intrinsic to hExo1 and are not dependent on the nature of the fluorophore.

    Techniques Used: Purification, Labeling, SDS Page, Electrophoretic Mobility Shift Assay, Activity Assay, Single-particle Tracking

    16) Product Images from "Modulating protein activity using tethered ligands with mutually exclusive binding sites"

    Article Title: Modulating protein activity using tethered ligands with mutually exclusive binding sites

    Journal: Nature Communications

    doi: 10.1038/ncomms8830

    Control of HCA by streptavidin. ( a ) Crystal structure of the active site of HCA bound to a benzenesulfonamide (PDB ID 1CNW). ( b ) Modulation of HCA activity in CLASH-Strep/HCA. A fusion protein of SNAP-tag, a 30-proline linker, NanoLuc Luciferase (NLuc) and HCA and is labelled with a synthetic molecule containing a fluorophore (red star) and the two intramolecular ligands benzenesulfonamide (SA) and biotin (B). Binding of streptavidin (Strep) to the tethered B displaces SA from HCA, leading both to a conformational change that can be seen by a decrease in BRET efficiency, and to an increase of enzymatic activity. ( c ) Chemical structure of the labelling compound. ( d ) Enzymatic activity of HCA and followed by measuring absorbance at 348 nm. Streptavidin (Strep) acts as an effector of the hydrolytic activity of HCA: in the absence of Strep, SA binds to and inhibits HCA (black dashed line), while on addition of saturation concentrations of Strep, the catalytic activity increases (black solid line). ( e ) Emission spectra of 10 nM CLASH-Strep/HCA at increasing streptavidin concentrations. ( f ) Titration of 10 nM CLASH-Strep/HCA with streptavidin. As a control, the synthetic ligand BG-Cy3-SA lacking the tethered biotin is used.
    Figure Legend Snippet: Control of HCA by streptavidin. ( a ) Crystal structure of the active site of HCA bound to a benzenesulfonamide (PDB ID 1CNW). ( b ) Modulation of HCA activity in CLASH-Strep/HCA. A fusion protein of SNAP-tag, a 30-proline linker, NanoLuc Luciferase (NLuc) and HCA and is labelled with a synthetic molecule containing a fluorophore (red star) and the two intramolecular ligands benzenesulfonamide (SA) and biotin (B). Binding of streptavidin (Strep) to the tethered B displaces SA from HCA, leading both to a conformational change that can be seen by a decrease in BRET efficiency, and to an increase of enzymatic activity. ( c ) Chemical structure of the labelling compound. ( d ) Enzymatic activity of HCA and followed by measuring absorbance at 348 nm. Streptavidin (Strep) acts as an effector of the hydrolytic activity of HCA: in the absence of Strep, SA binds to and inhibits HCA (black dashed line), while on addition of saturation concentrations of Strep, the catalytic activity increases (black solid line). ( e ) Emission spectra of 10 nM CLASH-Strep/HCA at increasing streptavidin concentrations. ( f ) Titration of 10 nM CLASH-Strep/HCA with streptavidin. As a control, the synthetic ligand BG-Cy3-SA lacking the tethered biotin is used.

    Techniques Used: High Content Screening, Activity Assay, Luciferase, Binding Assay, Bioluminescence Resonance Energy Transfer, Titration

    Control of a luciferase. ( a ) Schematic principle of the regulation of a luciferase by an exogenous effector: the dual ligand coelenteramide/biotin can bind to luciferase (Luc) or streptavidin (Strep) separately but not to both at the same time. Binding of streptavidin to the secondary ligand biotin (B) makes the tethered coelenteramide (C) unbind from the luciferase active site, leading to a change in bioluminescence. ( b ) Crystal structures of the active sites of Renilla luciferase with the inhibitor coelenteramide (blue, PDB ID 2PSJ) and of streptavidin bound to biotin (green, PDB ID 3RY2). ( c ) Structure of the dual ligand labelling molecule. ( d ) Luminescence intensity of 100 μl of 25 nM CLASH-Strep/Luc mixed with 2.5 μg ml −1 of the substrate coelenterazine in absence and in presence of 1 μM streptavidin. ( e ) Picture of the same solutions in transparent tubes, taken with a Canon 600D camera.
    Figure Legend Snippet: Control of a luciferase. ( a ) Schematic principle of the regulation of a luciferase by an exogenous effector: the dual ligand coelenteramide/biotin can bind to luciferase (Luc) or streptavidin (Strep) separately but not to both at the same time. Binding of streptavidin to the secondary ligand biotin (B) makes the tethered coelenteramide (C) unbind from the luciferase active site, leading to a change in bioluminescence. ( b ) Crystal structures of the active sites of Renilla luciferase with the inhibitor coelenteramide (blue, PDB ID 2PSJ) and of streptavidin bound to biotin (green, PDB ID 3RY2). ( c ) Structure of the dual ligand labelling molecule. ( d ) Luminescence intensity of 100 μl of 25 nM CLASH-Strep/Luc mixed with 2.5 μg ml −1 of the substrate coelenterazine in absence and in presence of 1 μM streptavidin. ( e ) Picture of the same solutions in transparent tubes, taken with a Canon 600D camera.

    Techniques Used: Luciferase, Binding Assay

    17) Product Images from "Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection"

    Article Title: Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s16081197

    Schematic illustration of the strategy for Hg 2+ ion detection using a DNA aptamer, a strand displacement reaction, and a label-free WGM sensor. Steps for performing the sensing experiments are: Step 1—attachment of cDNA via streptavidin to surface-adsorbed biotinylated dextran layer; for details of this functionalization procedure see Figure 3 a. Wash with buffer. Step 2—hybridization of aptamer, also see Figure 3 b for details. Wash with buffer. Step 3—detection of Hg 2+ by strand displacement reaction.
    Figure Legend Snippet: Schematic illustration of the strategy for Hg 2+ ion detection using a DNA aptamer, a strand displacement reaction, and a label-free WGM sensor. Steps for performing the sensing experiments are: Step 1—attachment of cDNA via streptavidin to surface-adsorbed biotinylated dextran layer; for details of this functionalization procedure see Figure 3 a. Wash with buffer. Step 2—hybridization of aptamer, also see Figure 3 b for details. Wash with buffer. Step 3—detection of Hg 2+ by strand displacement reaction.

    Techniques Used: Hybridization

    Functionalizing the microsphere for DNA strand displacement reaction. ( a ) WGM mass-loading curve recorded for streptavidin-cDNA complex binding to previously biotin-dextran–modified microsphere; ( b ) WGM mass-loading curve recorded for subsequent hybridization of Hg 2+ aptamer with cDNA at the microsphere surface. The three curves correspond to three independent experiments performed with different microspheres.
    Figure Legend Snippet: Functionalizing the microsphere for DNA strand displacement reaction. ( a ) WGM mass-loading curve recorded for streptavidin-cDNA complex binding to previously biotin-dextran–modified microsphere; ( b ) WGM mass-loading curve recorded for subsequent hybridization of Hg 2+ aptamer with cDNA at the microsphere surface. The three curves correspond to three independent experiments performed with different microspheres.

    Techniques Used: Binding Assay, Modification, Hybridization

    18) Product Images from "Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event"

    Article Title: Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2015.08.005

    Nucleosome disassembly. (A) A radiolabeled PCNA probe is loaded onto a linear DNA template, flanked by a single nucleosome at one extremity and a biotin-streptavidin block at the other. DNA flows into the exclusion volume when applied onto a gel filtration
    Figure Legend Snippet: Nucleosome disassembly. (A) A radiolabeled PCNA probe is loaded onto a linear DNA template, flanked by a single nucleosome at one extremity and a biotin-streptavidin block at the other. DNA flows into the exclusion volume when applied onto a gel filtration

    Techniques Used: Blocking Assay, Filtration

    19) Product Images from "Designed Auto-assembly of Nanostreptabodies for Rapid Tissue-specific Targeting in Vivo *"

    Article Title: Designed Auto-assembly of Nanostreptabodies for Rapid Tissue-specific Targeting in Vivo *

    Journal:

    doi: 10.1074/jbc.M109.061838

    Schematic representation of streptavidin/antibody complex formation. Bisbiotinylated mAbs react with SA (or avidin) to give the monoSA/antibody complex I and the monoSA/bisantibody complex II ; monobiotinylated Fabs react with SA to give streptabodies
    Figure Legend Snippet: Schematic representation of streptavidin/antibody complex formation. Bisbiotinylated mAbs react with SA (or avidin) to give the monoSA/antibody complex I and the monoSA/bisantibody complex II ; monobiotinylated Fabs react with SA to give streptabodies

    Techniques Used: Avidin-Biotin Assay

    20) Product Images from "Protection of Nonself Surfaces from Complement Attack by Factor H-Binding Peptides: Implications for Therapeutic Medicine"

    Article Title: Protection of Nonself Surfaces from Complement Attack by Factor H-Binding Peptides: Implications for Therapeutic Medicine

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1003802

    Binding of factor H to surfaces coated with synthetic 5C6 peptide as assessed by ELISA ( A ) and SPR ( B , C ). A , Polystyrene wells were coated with streptavidin, saturated with BSA, and incubated with biotinylated 5C6 peptide or negative control peptide;
    Figure Legend Snippet: Binding of factor H to surfaces coated with synthetic 5C6 peptide as assessed by ELISA ( A ) and SPR ( B , C ). A , Polystyrene wells were coated with streptavidin, saturated with BSA, and incubated with biotinylated 5C6 peptide or negative control peptide;

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Incubation, Negative Control

    Inhibition of complement activation by immobilized 5C6 on a polystyrene surface carrying an HSA layer. Biotinylated 5C6 and the control peptide (linear compstatin, LC) were captured by coated streptavidin. After being saturated with HSA, wells were incubated
    Figure Legend Snippet: Inhibition of complement activation by immobilized 5C6 on a polystyrene surface carrying an HSA layer. Biotinylated 5C6 and the control peptide (linear compstatin, LC) were captured by coated streptavidin. After being saturated with HSA, wells were incubated

    Techniques Used: Inhibition, Activation Assay, Incubation

    Inhibition of complement activation by immobilized 5C6 peptide on a model polystyrene surface in absence of an initial plasma protein layer. Biotinylated 5C6 peptide and the negative control peptide (linear compstatin, LC) were captured by coated streptavidin.
    Figure Legend Snippet: Inhibition of complement activation by immobilized 5C6 peptide on a model polystyrene surface in absence of an initial plasma protein layer. Biotinylated 5C6 peptide and the negative control peptide (linear compstatin, LC) were captured by coated streptavidin.

    Techniques Used: Inhibition, Activation Assay, Negative Control

    Related Articles

    Centrifugation:

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer. .. Expression was carried out in 293Freestyle media for 6 days with gentle shaking at 37 °C in the presence of 5% CO2 after which cells and cellular debris were removed by centrifugation at 10,000g followed by filtration through a 0.2 μM filter.

    Amplification:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: Briefly: the Spinach2-Streptavidin biosensor sequence was PCR amplified with a primer set to add an upstream T7 promoter for in vitro transcription. .. 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ).

    Filtration:

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer. .. Expression was carried out in 293Freestyle media for 6 days with gentle shaking at 37 °C in the presence of 5% CO2 after which cells and cellular debris were removed by centrifugation at 10,000g followed by filtration through a 0.2 μM filter.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction
    Article Snippet: Paragraph title: 2.4. Enzyme-linked immunosorbent assay for immobilization analysis ... High-binding 96-well polystyrene plates (Corning) were coated with streptavidin (NEB), as previously described [ ].

    Article Title: T7 RNA polymerase as a self-replicating label for antigen quantification
    Article Snippet: .. For comparison of detectabilities, we also performed a classical ELISA assay in which the immunocomplex was detected by using a streptavidin–alkaline phosphatase conjugate (New England BioLabs) and the enzymic activity was determined by adding p -nitrophenylphosphate as a substrate (Sigma) and measuring the absorbance at 405 nm on a microplate photometer (model EL-307C; BioTek Instruments, Winooski, VT). ..

    Incubation:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ). .. Each sample (standard curve point or re-suspended sample) was mixed with a solution of 10 μM Spinach2-Streptavidin aptamer and 2 μM DFHBI-1T (Lucerna, Inc.) and incubated at 25 °C for 14 h in a buffer containing 100 mM Tris, pH 7.5, 200 mM KCl, 10 mM NaCl and 1 mM MgCl2 .

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction
    Article Snippet: High-binding 96-well polystyrene plates (Corning) were coated with streptavidin (NEB), as previously described [ ]. .. After 45 min of incubation, unbound scFvs were washed off with PBST, and the immobilized scFvs were incubated for 1 h with an anti-FLAG antibody (HRP-conjugated, Sigma).

    Article Title: Proteins mediating DNA loops effectively block transcription
    Article Snippet: .. Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min. .. This droplet was rinsed with 400 μl of high‐performance liquid chromatography grade water and dried gently with compressed air.

    Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *
    Article Snippet: The streptavidin-DNA complex was generated from 500 fmol of biotinylated DNA (25 n m ) mixed with 5 pmol of streptavidin (250 n m ) (New England Biolabs) and allowed to bind for 1 h at 4 °C. .. 10 picomoles of NPH I (500 n m ) were added to the reactions and incubated at 30 °C, and samples were removed at various times.

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: Probes were attached to streptavidin (New England Biolabs (NEB)) in a 20 μl reaction containing 25 nM streptavidin (NEB) and 140 nM probe (for a total of more than 4:1 probe/streptavidin to ensure streptavidin saturation), held at 37 °C for 1 h. Other probes were covalently linked to alkyne-modified strands by click chemistry. .. A volume of 9 μl of 1 mM DBCO-linker strands and 1 μl of 10× phosphate-buffered saline (pH 7.4) buffer were added to 20 μl of water containing 180 pmol azide-modified probes and incubated at room temperature for 12 h. The linked probes were purified by 8% denaturing polyacrylamide gel and quantified by NanoDrop UV–Vis Spectrophotometer (Thermo Scientific).

    Luciferase:

    Article Title: T7 RNA polymerase as a self-replicating label for antigen quantification
    Article Snippet: For comparison of detectabilities, we also performed a classical ELISA assay in which the immunocomplex was detected by using a streptavidin–alkaline phosphatase conjugate (New England BioLabs) and the enzymic activity was determined by adding p -nitrophenylphosphate as a substrate (Sigma) and measuring the absorbance at 405 nm on a microplate photometer (model EL-307C; BioTek Instruments, Winooski, VT). .. To assess the reproducibility of the proposed immunoassay (including all the steps, i.e. coating of the wells, immunocomplex formation, binding of SA–BT7RP, in vitro coupled transcription/translation and luciferase measurement), we analyzed samples containing 0.1, 1 and 10 fmol PSA.

    Activity Assay:

    Article Title: T7 RNA polymerase as a self-replicating label for antigen quantification
    Article Snippet: .. For comparison of detectabilities, we also performed a classical ELISA assay in which the immunocomplex was detected by using a streptavidin–alkaline phosphatase conjugate (New England BioLabs) and the enzymic activity was determined by adding p -nitrophenylphosphate as a substrate (Sigma) and measuring the absorbance at 405 nm on a microplate photometer (model EL-307C; BioTek Instruments, Winooski, VT). ..

    Expressing:

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer. .. Expression was carried out in 293Freestyle media for 6 days with gentle shaking at 37 °C in the presence of 5% CO2 after which cells and cellular debris were removed by centrifugation at 10,000g followed by filtration through a 0.2 μM filter.

    Article Title: T7 RNA polymerase as a self-replicating label for antigen quantification
    Article Snippet: When the self-replication system was used (single expression reaction with a delayed addition of Luc-DNA), as low as 12 amol PSA could be detected with a S/B ratio of 1.9. .. For comparison of detectabilities, we also performed a classical ELISA assay in which the immunocomplex was detected by using a streptavidin–alkaline phosphatase conjugate (New England BioLabs) and the enzymic activity was determined by adding p -nitrophenylphosphate as a substrate (Sigma) and measuring the absorbance at 405 nm on a microplate photometer (model EL-307C; BioTek Instruments, Winooski, VT).

    Modification:

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: Purified biotinylated-avi tagged 426cTM4ΔV1-3 gp120 or gp140 was mixed with a biotinylated dextran (Life Technologies, Cat # D-7142) at a 3:1 ratio (Env: biotin), with the assumption that the modified dextran had 77 biotins molecules per multimer (lot-dependent value). .. Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer.

    Article Title: Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures
    Article Snippet: Prior to the electrical test with streptavidin, we performed a control experiment by placing a 1 μL drop of d-biotin-saturated streptavidin (prepared by adding four equivalents of d-biotin (Supelco, Bellefonte, PA) to one equivalent of streptavidin in 1 mM phosphate buffer) on the BAC-BSA modified sensor and measured the conductance. .. Following the control experiment we rinsed the sensor with buffer solution and placed a 1 μL drop of 500 nM streptavidin (New England BioLabs, Ipswich, MA) on the sensor and repeated the conductance measurement.

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: Some probes were ordered with an internal biotin modification in the center of the 5 nt loop domain, in which case probes were held in proximity with streptavidin, as indicated in Fig. and Supplementary Fig. . .. Probes were attached to streptavidin (New England Biolabs (NEB)) in a 20 μl reaction containing 25 nM streptavidin (NEB) and 140 nM probe (for a total of more than 4:1 probe/streptavidin to ensure streptavidin saturation), held at 37 °C for 1 h. Other probes were covalently linked to alkyne-modified strands by click chemistry.

    High Performance Liquid Chromatography:

    Article Title: Proteins mediating DNA loops effectively block transcription
    Article Snippet: The poly‐L‐ornithine‐coated mica was rinsed drop‐wise with 400 μl of high‐performance liquid chromatography grade water and dried with compressed air. .. Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min.

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: Strands were ordered at 100 or 250 nmol scale, with high-performance liquid chromatography (HPLC) purification. .. Probes were attached to streptavidin (New England Biolabs (NEB)) in a 20 μl reaction containing 25 nM streptavidin (NEB) and 140 nM probe (for a total of more than 4:1 probe/streptavidin to ensure streptavidin saturation), held at 37 °C for 1 h. Other probes were covalently linked to alkyne-modified strands by click chemistry.

    Transfection:

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: .. Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer. .. Expression was carried out in 293Freestyle media for 6 days with gentle shaking at 37 °C in the presence of 5% CO2 after which cells and cellular debris were removed by centrifugation at 10,000g followed by filtration through a 0.2 μM filter.

    Transferring:

    Article Title: Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures
    Article Snippet: Following the control experiment we rinsed the sensor with buffer solution and placed a 1 μL drop of 500 nM streptavidin (New England BioLabs, Ipswich, MA) on the sensor and repeated the conductance measurement. .. As in the DNA experimental procedure, we added all solutions directly to the sensor structure using a pipette while the sensor remained fixed in a probe station.

    Generated:

    Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *
    Article Snippet: .. The streptavidin-DNA complex was generated from 500 fmol of biotinylated DNA (25 n m ) mixed with 5 pmol of streptavidin (250 n m ) (New England Biolabs) and allowed to bind for 1 h at 4 °C. .. The streptavidin-bound DNA in 20-μl total volume containing 20 m m Tris-HCl (pH 8.0), 6 m m MgCl2 , 2 m m DTT, and 8% glycerol was mixed with 100 pmol of biotin (5 μ m ) and 1 m m ATP.

    other:

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation
    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Sequencing:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: Briefly: the Spinach2-Streptavidin biosensor sequence was PCR amplified with a primer set to add an upstream T7 promoter for in vitro transcription. .. 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ).

    Binding Assay:

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer. .. Clarified supernatant was passed over a GNL agarose column (Vector Laboratories) pre-equilibrated in GNL binding buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4) followed by extensive washing and then eluted with GNL binding buffer containing 1 M methylmannopyranoside.

    Article Title: Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures
    Article Snippet: This control experiment relied on the fact that the streptavidin to which the sensor was exposed had its biotin binding sites occupied by d-biotin, preventing it from binding to the biotin on the sensor surface. .. Following the control experiment we rinsed the sensor with buffer solution and placed a 1 μL drop of 500 nM streptavidin (New England BioLabs, Ipswich, MA) on the sensor and repeated the conductance measurement.

    Article Title: T7 RNA polymerase as a self-replicating label for antigen quantification
    Article Snippet: For comparison of detectabilities, we also performed a classical ELISA assay in which the immunocomplex was detected by using a streptavidin–alkaline phosphatase conjugate (New England BioLabs) and the enzymic activity was determined by adding p -nitrophenylphosphate as a substrate (Sigma) and measuring the absorbance at 405 nm on a microplate photometer (model EL-307C; BioTek Instruments, Winooski, VT). .. To assess the reproducibility of the proposed immunoassay (including all the steps, i.e. coating of the wells, immunocomplex formation, binding of SA–BT7RP, in vitro coupled transcription/translation and luciferase measurement), we analyzed samples containing 0.1, 1 and 10 fmol PSA.

    Nucleic Acid Electrophoresis:

    Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *
    Article Snippet: Paragraph title: Analysis of NPH I Streptavidin Displacement Assays by Gel Electrophoresis ... The streptavidin-DNA complex was generated from 500 fmol of biotinylated DNA (25 n m ) mixed with 5 pmol of streptavidin (250 n m ) (New England Biolabs) and allowed to bind for 1 h at 4 °C.

    Fluorescence:

    Article Title: Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures
    Article Snippet: We then rinsed the chip with DI water and compared the fluorescence image of this wafer with one that underwent the same process but without the BAC-BSA modification. .. Following the control experiment we rinsed the sensor with buffer solution and placed a 1 μL drop of 500 nM streptavidin (New England BioLabs, Ipswich, MA) on the sensor and repeated the conductance measurement.

    Isolation:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: Pure RNA was isolated by standard ethanol precipitation. .. 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ).

    Flow Cytometry:

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation
    Article Snippet: .. When a higher streptavidin concentration (10 μg/mL) was introduced into a biotin-modified channel , more rapid cross-linking of the biotin anchored to the surface in the first few millimeters of the channel length led to faster constriction at the start of the channel and a shorter capillary flow distance traveled by the streptavidin solution. .. On the other hand, when a lower concentration of streptavidin solution (100 ng/mL) was loaded , slower cross-linking led to a greater capillary flow distance for the streptavidin solution before constriction in the first few millimeters of the channel stopped flow.

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation
    Article Snippet: Finally, PBS was aspirated from the channel and a 1 μL streptavidin solution (New England Biolabs, Ipswich, MA) of specified concentration in PBS was pipetted into the reservoir (see ). .. The flow distance of streptavidin solution in the microchannel was measured with a ruler, and photographs were obtained with a digital camera.

    Microscopy:

    Article Title: Proteins mediating DNA loops effectively block transcription
    Article Snippet: Paragraph title: Sample preparation for scanning force microscopy ... Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min.

    Size-exclusion Chromatography:

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer. .. Ferritin nanoparticles were further purified by SEC using a 16/60 S200 column (GE Healthcare) equilibrated in PBS, followed by a second final SEC purification step on a 10/300 superose 6 column equilibrated in PBS (GE Healthcare).

    Polymerase Chain Reaction:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: The resulting PCR product was purified by spin column (Thermo Scientific) and used as a template for in vitro T7 transcription using the AmpliScribe T7-Flash Transcription kit (Epicentre) according to the manufacturer protocol. .. 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ).

    Purification:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: The resulting PCR product was purified by spin column (Thermo Scientific) and used as a template for in vitro T7 transcription using the AmpliScribe T7-Flash Transcription kit (Epicentre) according to the manufacturer protocol. .. 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ).

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: Purified biotinylated-avi tagged 426cTM4ΔV1-3 gp120 or gp140 was mixed with a biotinylated dextran (Life Technologies, Cat # D-7142) at a 3:1 ratio (Env: biotin), with the assumption that the modified dextran had 77 biotins molecules per multimer (lot-dependent value). .. Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer.

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: Strands were ordered at 100 or 250 nmol scale, with high-performance liquid chromatography (HPLC) purification. .. Probes were attached to streptavidin (New England Biolabs (NEB)) in a 20 μl reaction containing 25 nM streptavidin (NEB) and 140 nM probe (for a total of more than 4:1 probe/streptavidin to ensure streptavidin saturation), held at 37 °C for 1 h. Other probes were covalently linked to alkyne-modified strands by click chemistry.

    Chromatin Immunoprecipitation:

    Article Title: Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures
    Article Snippet: We then rinsed the chip with DI water and compared the fluorescence image of this wafer with one that underwent the same process but without the BAC-BSA modification. .. Following the control experiment we rinsed the sensor with buffer solution and placed a 1 μL drop of 500 nM streptavidin (New England BioLabs, Ipswich, MA) on the sensor and repeated the conductance measurement.

    Plasmid Preparation:

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice
    Article Snippet: Streptavidin (New England Biolabs Cat# N7021S) was then added to achieve a 3:1:1 Env to streptavidin to biotin ratio. pTT3-426cTM4ΔV1-3-ferritin was transfected into 293E cells at a density of 106 cells ml−1 in Freestyle 293 media (Life Technologies) using the 293Free transfection reagent (EMD Millipore) and half the amount of DNA recommended by the manufacturer. .. Clarified supernatant was passed over a GNL agarose column (Vector Laboratories) pre-equilibrated in GNL binding buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4) followed by extensive washing and then eluted with GNL binding buffer containing 1 M methylmannopyranoside.

    Sample Prep:

    Article Title: Proteins mediating DNA loops effectively block transcription
    Article Snippet: Paragraph title: Sample preparation for scanning force microscopy ... Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min.

    In Vitro:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: The resulting PCR product was purified by spin column (Thermo Scientific) and used as a template for in vitro T7 transcription using the AmpliScribe T7-Flash Transcription kit (Epicentre) according to the manufacturer protocol. .. 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ).

    Article Title: T7 RNA polymerase as a self-replicating label for antigen quantification
    Article Snippet: For comparison of detectabilities, we also performed a classical ELISA assay in which the immunocomplex was detected by using a streptavidin–alkaline phosphatase conjugate (New England BioLabs) and the enzymic activity was determined by adding p -nitrophenylphosphate as a substrate (Sigma) and measuring the absorbance at 405 nm on a microplate photometer (model EL-307C; BioTek Instruments, Winooski, VT). .. To assess the reproducibility of the proposed immunoassay (including all the steps, i.e. coating of the wells, immunocomplex formation, binding of SA–BT7RP, in vitro coupled transcription/translation and luciferase measurement), we analyzed samples containing 0.1, 1 and 10 fmol PSA.

    Ethanol Precipitation:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: Pure RNA was isolated by standard ethanol precipitation. .. 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ).

    Spectrophotometry:

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: Probes were attached to streptavidin (New England Biolabs (NEB)) in a 20 μl reaction containing 25 nM streptavidin (NEB) and 140 nM probe (for a total of more than 4:1 probe/streptavidin to ensure streptavidin saturation), held at 37 °C for 1 h. Other probes were covalently linked to alkyne-modified strands by click chemistry. .. A volume of 9 μl of 1 mM DBCO-linker strands and 1 μl of 10× phosphate-buffered saline (pH 7.4) buffer were added to 20 μl of water containing 180 pmol azide-modified probes and incubated at room temperature for 12 h. The linked probes were purified by 8% denaturing polyacrylamide gel and quantified by NanoDrop UV–Vis Spectrophotometer (Thermo Scientific).

    Produced:

    Article Title: Proteins mediating DNA loops effectively block transcription
    Article Snippet: .. Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min. .. This droplet was rinsed with 400 μl of high‐performance liquid chromatography grade water and dried gently with compressed air.

    Concentration Assay:

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes
    Article Snippet: 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ). .. To make a standard curve sample, streptavidin stock (100 mg l−1 ) was diluted directly in 1× DPBS (Corning) to the target concentration.

    Article Title: Proteins mediating DNA loops effectively block transcription
    Article Snippet: .. Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min. .. This droplet was rinsed with 400 μl of high‐performance liquid chromatography grade water and dried gently with compressed air.

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation
    Article Snippet: .. When a higher streptavidin concentration (10 μg/mL) was introduced into a biotin-modified channel , more rapid cross-linking of the biotin anchored to the surface in the first few millimeters of the channel length led to faster constriction at the start of the channel and a shorter capillary flow distance traveled by the streptavidin solution. .. On the other hand, when a lower concentration of streptavidin solution (100 ng/mL) was loaded , slower cross-linking led to a greater capillary flow distance for the streptavidin solution before constriction in the first few millimeters of the channel stopped flow.

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation
    Article Snippet: .. Finally, PBS was aspirated from the channel and a 1 μL streptavidin solution (New England Biolabs, Ipswich, MA) of specified concentration in PBS was pipetted into the reservoir (see ). .. The flow distance of streptavidin solution in the microchannel was measured with a ruler, and photographs were obtained with a digital camera.

    BAC Assay:

    Article Title: Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures
    Article Snippet: Prior to the electrical test with streptavidin, we performed a control experiment by placing a 1 μL drop of d-biotin-saturated streptavidin (prepared by adding four equivalents of d-biotin (Supelco, Bellefonte, PA) to one equivalent of streptavidin in 1 mM phosphate buffer) on the BAC-BSA modified sensor and measured the conductance. .. Following the control experiment we rinsed the sensor with buffer solution and placed a 1 μL drop of 500 nM streptavidin (New England BioLabs, Ipswich, MA) on the sensor and repeated the conductance measurement.

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    New England Biolabs streptavidin solution
    Flow distance traveled as a function of <t>streptavidin</t> concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.
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    Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Journal: Analytical chemistry

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    doi: 10.1021/ac301278s

    Figure Lengend Snippet: Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Techniques: Flow Cytometry, Concentration Assay, Modification

    Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Journal: Analytical chemistry

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    doi: 10.1021/ac301278s

    Figure Lengend Snippet: Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Techniques: Flow Cytometry, Modification

    Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Journal: Analytical chemistry

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    doi: 10.1021/ac301278s

    Figure Lengend Snippet: Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Techniques: Flow Cytometry, Modification

    Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Journal: Analytical chemistry

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    doi: 10.1021/ac301278s

    Figure Lengend Snippet: Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Techniques: Fluorescence, Modification, Flow Cytometry

    Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.

    Journal: Biotechnology and applied biochemistry

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction

    doi: 10.1002/bab.1645

    Figure Lengend Snippet: Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.

    Article Snippet: High-binding 96-well polystyrene plates (Corning) were coated with streptavidin (NEB), as previously described [ ].

    Techniques: Construct, Concentration Assay

    Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.

    Journal: Biotechnology and applied biochemistry

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction

    doi: 10.1002/bab.1645

    Figure Lengend Snippet: Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.

    Article Snippet: High-binding 96-well polystyrene plates (Corning) were coated with streptavidin (NEB), as previously described [ ].

    Techniques: Construct, Concentration Assay

    RAPID screening for the improvement of streptavidin protein secretion through an evolved secretory tag. The RAPID screening approach was used to identify mutations in the α-mating factor (αMF) secretory leader fused to streptavidin in yeast. a Histograms of droplet fluorescence pre-sort and post-sort (re-encapsulated) demonstrate enrichment through the process. b Isolated and re-transformed clones randomly selected from the pre- and post-sort populations were quantified for streptavidin production. Error bars represent 95% confidence intervals of biological triplicates. Protein secretion from an individual clone was increased nearly threefold over wild-type production using this approach, and there was also twofold increase in secretion between the mean of the sorted and unsorted clones

    Journal: Nature Communications

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes

    doi: 10.1038/s41467-017-00425-7

    Figure Lengend Snippet: RAPID screening for the improvement of streptavidin protein secretion through an evolved secretory tag. The RAPID screening approach was used to identify mutations in the α-mating factor (αMF) secretory leader fused to streptavidin in yeast. a Histograms of droplet fluorescence pre-sort and post-sort (re-encapsulated) demonstrate enrichment through the process. b Isolated and re-transformed clones randomly selected from the pre- and post-sort populations were quantified for streptavidin production. Error bars represent 95% confidence intervals of biological triplicates. Protein secretion from an individual clone was increased nearly threefold over wild-type production using this approach, and there was also twofold increase in secretion between the mean of the sorted and unsorted clones

    Article Snippet: 1, 5, 10, 20, 30, 40, 50, 60 and 100 mg l−1 of streptavidin standard (NEB) were chosen as standard curve points (Supplementary Fig. ).

    Techniques: Fluorescence, Isolation, Transformation Assay, Clone Assay

    LacI bound to an O1 operator pauses transcription. (A) A schematic representation of the DNA template used in magnetic tweezer transcription assays. The template contained a T7A1 promoter close to the upstream end, a stall site at position +22, an O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. A streptavidin‐labeled paramagnetic bead was coupled to the biotin label to for micromanipulation in the magnetic tweezer. Four examples of transcriptional elongation recorded using the magnetic tweezers are displayed. In ( B ) no LacI was included and transcription shortened the DNA tether progressively without interruption. When LacI was included ( C–E ) , transcription shortened the tether by about 0.2 um before pausing for about 200 s and then resuming. Transcription finally ceased after the tether shortened by either 0.35 um (C and D), a distance corresponding to the location of a terminator sequence, or 0.5 um (B and E), a distance corresponding to the end of the template.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Proteins mediating DNA loops effectively block transcription

    doi: 10.1002/pro.3156

    Figure Lengend Snippet: LacI bound to an O1 operator pauses transcription. (A) A schematic representation of the DNA template used in magnetic tweezer transcription assays. The template contained a T7A1 promoter close to the upstream end, a stall site at position +22, an O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. A streptavidin‐labeled paramagnetic bead was coupled to the biotin label to for micromanipulation in the magnetic tweezer. Four examples of transcriptional elongation recorded using the magnetic tweezers are displayed. In ( B ) no LacI was included and transcription shortened the DNA tether progressively without interruption. When LacI was included ( C–E ) , transcription shortened the tether by about 0.2 um before pausing for about 200 s and then resuming. Transcription finally ceased after the tether shortened by either 0.35 um (C and D), a distance corresponding to the location of a terminator sequence, or 0.5 um (B and E), a distance corresponding to the end of the template.

    Article Snippet: Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min.

    Techniques: Labeling, Micromanipulation, Sequencing

    Nanographs of RNA polymerases trapped by EDTA quenching during elongation along DNA with and without LacI‐mediated loops. (A) Schematic representation of the DNA templates used in scanning force microscopy assays. All templates contained a T7A1 promoter close to the upstream end, a stall site at position +22, a “far” O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. The two DNA templates used for SFM measurements of transcription differed in the “near” operator positioned 253 bp downstream from the promoter; one template contained the Os operator while the other contained the O2 operator. The terminator was the very last feature of the sequence and was biotin labeled. Streptavidin was coupled to the biotin label to facilitate identifying the “downstream” end of the molecule in SFM nanographs. (B) The upper row is a selection of molecules along which RNA polymerases (large yellow particle) had not progressed very far from the transcription start site near the end of the DNA without a streptavidin particle (blue). Closed and open conformations of the LacI tetramers are visible for either looped (left) or unlooped (right) columns. The closed conformations are shown as blue particles that are slightly larger than the streptavidin. In the open conformation, two lobes are visible especially on looped DNA. These lobes correspond to individual dimers with DNA binding head groups. The TECs shown in the lower row had progressed further and small coils of RNA emanate from them (see inset schematics for the regions of interest). These TECs have collided with LacI particles. The LacI particles correspond to blue protuberances on the periphery of the larger yellow RNA polymerase particle. The RNA polymerases themselves appear to shift to the side opposite LacI especially for open LacI conformations.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Proteins mediating DNA loops effectively block transcription

    doi: 10.1002/pro.3156

    Figure Lengend Snippet: Nanographs of RNA polymerases trapped by EDTA quenching during elongation along DNA with and without LacI‐mediated loops. (A) Schematic representation of the DNA templates used in scanning force microscopy assays. All templates contained a T7A1 promoter close to the upstream end, a stall site at position +22, a “far” O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. The two DNA templates used for SFM measurements of transcription differed in the “near” operator positioned 253 bp downstream from the promoter; one template contained the Os operator while the other contained the O2 operator. The terminator was the very last feature of the sequence and was biotin labeled. Streptavidin was coupled to the biotin label to facilitate identifying the “downstream” end of the molecule in SFM nanographs. (B) The upper row is a selection of molecules along which RNA polymerases (large yellow particle) had not progressed very far from the transcription start site near the end of the DNA without a streptavidin particle (blue). Closed and open conformations of the LacI tetramers are visible for either looped (left) or unlooped (right) columns. The closed conformations are shown as blue particles that are slightly larger than the streptavidin. In the open conformation, two lobes are visible especially on looped DNA. These lobes correspond to individual dimers with DNA binding head groups. The TECs shown in the lower row had progressed further and small coils of RNA emanate from them (see inset schematics for the regions of interest). These TECs have collided with LacI particles. The LacI particles correspond to blue protuberances on the periphery of the larger yellow RNA polymerase particle. The RNA polymerases themselves appear to shift to the side opposite LacI especially for open LacI conformations.

    Article Snippet: Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min.

    Techniques: Microscopy, Sequencing, Labeling, Selection, Binding Assay

    Nanographs of RNAP elongation along DNA with and without LacI‐mediated loops. (Top) Schematic representations of transcription elongation complexes (TECs). Columns correspond to different DNA topologies and LacI occupancy. The first column corresponds to transcription elongation without LacI in the reaction buffer. The second and third column correspond to DNA found in a looped topology, with LacI at each of the two operators; the fourth and fifth columns correspond to unlooped DNA with both operators occupied. Transcription elongation progress is categorized in five zones (roman numerals I‐V). Numerals in the schematic correspond to each row of the nanograph array. Each image in the array is representative of its corresponding category (columns) and elongation progress (rows). Image colors indicate height, according to the color scale below. RNAP, LacI and streptavidin particles are indicated by yellow, light blue, and white arrows respectively. (Row I) AFM images of RNAP bound at the T7A1 promoter. (Row II) Images in which TECs have not yet reached the near operator. (Row III) Images in which TECs contact LacI at the near operator. (Row IV) Images in which TECs were found between the two operators. (Row V) Images in which TECs were beyond the far operator. As indicated in the figure, images for RNAP in zone V were not detected for looped O2‐O1 DNA and unlooped Os‐O1 DNA. Note that nascent RNA associated with each TEC is visible, especially in the first column (insets), and increases in size as the RNAP progresses (I to V).

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Proteins mediating DNA loops effectively block transcription

    doi: 10.1002/pro.3156

    Figure Lengend Snippet: Nanographs of RNAP elongation along DNA with and without LacI‐mediated loops. (Top) Schematic representations of transcription elongation complexes (TECs). Columns correspond to different DNA topologies and LacI occupancy. The first column corresponds to transcription elongation without LacI in the reaction buffer. The second and third column correspond to DNA found in a looped topology, with LacI at each of the two operators; the fourth and fifth columns correspond to unlooped DNA with both operators occupied. Transcription elongation progress is categorized in five zones (roman numerals I‐V). Numerals in the schematic correspond to each row of the nanograph array. Each image in the array is representative of its corresponding category (columns) and elongation progress (rows). Image colors indicate height, according to the color scale below. RNAP, LacI and streptavidin particles are indicated by yellow, light blue, and white arrows respectively. (Row I) AFM images of RNAP bound at the T7A1 promoter. (Row II) Images in which TECs have not yet reached the near operator. (Row III) Images in which TECs contact LacI at the near operator. (Row IV) Images in which TECs were found between the two operators. (Row V) Images in which TECs were beyond the far operator. As indicated in the figure, images for RNAP in zone V were not detected for looped O2‐O1 DNA and unlooped Os‐O1 DNA. Note that nascent RNA associated with each TEC is visible, especially in the first column (insets), and increases in size as the RNAP progresses (I to V).

    Article Snippet: Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min.

    Techniques: