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    Thermo Fisher streptavidin horseradish
    Streptavidin Horseradish, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Article Title: Immune response development after vaccination of 1-day-old naïve pigs with a Porcine Reproductive and Respiratory Syndrome 1-based modified live virus vaccine
    Article Snippet: The cut-off point of each ELISA was calculated as the mean + 3SD OD (optical density) of negative controls. .. Positive reactions were revealed using Streptavidin-Horseradish (Thermo Fisher Scientific) and soluble TMB (Merck Millipore).

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    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1000666

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

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

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

    Characterization of TurboID and miniTurbo in mammalian cells ( a ) Comparison of TurboID and miniTurbo to three other promiscuous ligases (BioID 5 , BioID2 26 , and BASU 27 ) in the cytosol of HEK 293T cells. Here, 500 μM exogenous biotin was used for labeling, whereas 50 μM was used in Supplementary Figure 6c-e . Streptavidin-HRP blotting detects promiscuously biotinylated proteins, and anti-V5 blotting detects ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation bands. This experiment was performed twice with similar results. ( b ) Quantitation of experiment in (a). For shorter timepoints (-biotin and 10 min), we used a longer-exposure image of the same blot, shown in Supplementary Figure 6a ; for longer timepoints (1, 6, 18 hr), we used a shorter-exposure image of the blot in (a), shown in Supplementary Figure 6b . Quantitation performed as in Figure 1g . Grey dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity calculated from the two replicates. ( c ) Comparison of promiscuous ligases in multiple HEK organelles. Each ligase was fused to a peptide targeting sequence (see Supplementary Table 8 ) directing them to the locations indicated in the scheme at right. BioID samples were treated with 50 μM biotin for 18 hours. TurboID and miniTurbo samples were labeled for 10 minutes with 50 (+) or 500 (++) μM biotin. U, untransfected. Asterisks denote ligase self-biotinylation. This experiment was performed five times for nuclear constructs, three for mitochondrial constructs, four times for ER membrane constructs, and twice for ER lumen constructs with similar results. ( d ) Mass spectrometry-based proteomic experiment comparing TurboID and BioID on the ER membrane (ERM), facing cytosol. Experimental design and labeling conditions. Ligase fusion constructs were stably expressed in HEK 239T. BioID samples were treated with 50 μM biotin for 18 hours, while TurboID samples were treated with 500 μM biotin for 10 minutes or 1 hr. After labeling, cells were lysed and biotinylated proteins were enriched with streptavidin beads, digested to peptides, and conjugated to TMT (tandem mass tag) labels. All 11 samples were then combined and analyzed by LC-MS/MS. This experiment was performed once with two replicates per condition. ( e ) Specificity analysis for proteomic datasets derived from experiment in (d). Size of each ERM proteome at top. Bars show percentage of each proteome with prior secretory pathway annotation, according to GOCC, Phobius, human protein atlas, human plasma proteome database, and literature (see Methods and Supplementary Table 2 Tab 4 ). ( f ) Same as (e), except for each ERM proteome, we analyze the subset with ER, Golgi, or plasma membrane annotation. Annotations from GOCC were assigned in the priority order: ER > Golgi > plasma membrane (see Methods and Supplementary Table 2 Tab 5 ). ( g ) Breakdown of ER proteins enriched by TurboID and BioID, by transmembrane or soluble. Soluble proteins were further divided into luminal or cytosol-facing. Annotations obtained from GOCC, UniProt, TMHMM, and literature (see Methods and Supplementary Table 2 Tab 6 ). ( h ) Characterization of nuclear and mitochondrial matrix proteomes obtained via BioID (18 hour), TurboID (10 min), and miniTurbo (10 min)-catalyzed labeling. Proteome sizes across top. Bars show fraction of each nuclear (left) or mitochondrial (right) proteome with prior nuclear or mitochondrial annotation, according to GOCC, MitoCarta, or literature (see Methods and Supplementary Table 3 Tab 1, Supplementary Table 4, Tab 1 ). Design of proteomic experiment shown in Supplementary Figure 10a , proteomic lists in Supplementary Tables 6-7 ; further analysis of proteome data in Supplementary Figure 10 .

    Journal: Nature biotechnology

    Article Title: Efficient proximity labeling in living cells and organisms with TurboID

    doi: 10.1038/nbt.4201

    Figure Lengend Snippet: Characterization of TurboID and miniTurbo in mammalian cells ( a ) Comparison of TurboID and miniTurbo to three other promiscuous ligases (BioID 5 , BioID2 26 , and BASU 27 ) in the cytosol of HEK 293T cells. Here, 500 μM exogenous biotin was used for labeling, whereas 50 μM was used in Supplementary Figure 6c-e . Streptavidin-HRP blotting detects promiscuously biotinylated proteins, and anti-V5 blotting detects ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation bands. This experiment was performed twice with similar results. ( b ) Quantitation of experiment in (a). For shorter timepoints (-biotin and 10 min), we used a longer-exposure image of the same blot, shown in Supplementary Figure 6a ; for longer timepoints (1, 6, 18 hr), we used a shorter-exposure image of the blot in (a), shown in Supplementary Figure 6b . Quantitation performed as in Figure 1g . Grey dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity calculated from the two replicates. ( c ) Comparison of promiscuous ligases in multiple HEK organelles. Each ligase was fused to a peptide targeting sequence (see Supplementary Table 8 ) directing them to the locations indicated in the scheme at right. BioID samples were treated with 50 μM biotin for 18 hours. TurboID and miniTurbo samples were labeled for 10 minutes with 50 (+) or 500 (++) μM biotin. U, untransfected. Asterisks denote ligase self-biotinylation. This experiment was performed five times for nuclear constructs, three for mitochondrial constructs, four times for ER membrane constructs, and twice for ER lumen constructs with similar results. ( d ) Mass spectrometry-based proteomic experiment comparing TurboID and BioID on the ER membrane (ERM), facing cytosol. Experimental design and labeling conditions. Ligase fusion constructs were stably expressed in HEK 239T. BioID samples were treated with 50 μM biotin for 18 hours, while TurboID samples were treated with 500 μM biotin for 10 minutes or 1 hr. After labeling, cells were lysed and biotinylated proteins were enriched with streptavidin beads, digested to peptides, and conjugated to TMT (tandem mass tag) labels. All 11 samples were then combined and analyzed by LC-MS/MS. This experiment was performed once with two replicates per condition. ( e ) Specificity analysis for proteomic datasets derived from experiment in (d). Size of each ERM proteome at top. Bars show percentage of each proteome with prior secretory pathway annotation, according to GOCC, Phobius, human protein atlas, human plasma proteome database, and literature (see Methods and Supplementary Table 2 Tab 4 ). ( f ) Same as (e), except for each ERM proteome, we analyze the subset with ER, Golgi, or plasma membrane annotation. Annotations from GOCC were assigned in the priority order: ER > Golgi > plasma membrane (see Methods and Supplementary Table 2 Tab 5 ). ( g ) Breakdown of ER proteins enriched by TurboID and BioID, by transmembrane or soluble. Soluble proteins were further divided into luminal or cytosol-facing. Annotations obtained from GOCC, UniProt, TMHMM, and literature (see Methods and Supplementary Table 2 Tab 6 ). ( h ) Characterization of nuclear and mitochondrial matrix proteomes obtained via BioID (18 hour), TurboID (10 min), and miniTurbo (10 min)-catalyzed labeling. Proteome sizes across top. Bars show fraction of each nuclear (left) or mitochondrial (right) proteome with prior nuclear or mitochondrial annotation, according to GOCC, MitoCarta, or literature (see Methods and Supplementary Table 3 Tab 1, Supplementary Table 4, Tab 1 ). Design of proteomic experiment shown in Supplementary Figure 10a , proteomic lists in Supplementary Tables 6-7 ; further analysis of proteome data in Supplementary Figure 10 .

    Article Snippet: To detect biotinylated proteins, blots were incubated with 0.3 μg/mL streptavidin-HRP (Thermo Fisher S911) in PBST-BSA for 1 hour at room temperature.

    Techniques: Labeling, Expressing, Quantitation Assay, Sequencing, Construct, Mass Spectrometry, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

    Directed evolution of TurboID ( a ) Proximity-dependent biotinylation catalyzed by promiscuous biotin ligases. Ligases catalyze the formation of biotin-5′-AMP anhydride, which diffuses out of the active site to biotinylate proximal endogenous proteins on nucleophilic residues such as lysine. ( b ) Yeast display-based selection scheme. A > 10 7 library of ligase variants is displayed on the yeast surface as a fusion to mating protein Aga2p. All ligases have a C-terminal myc epitope tag. Biotin and ATP are added to the yeast library for between 10 minutes and 24 hours. Ligase-catalyzed promiscuous biotinylation is detected by staining with streptavidin-phycoerythrin and ligase expression is detected by staining with anti-myc antibody. Two-dimensional FACS sorting enables enrichment of cells displaying a high ratio of streptavidin to myc staining. ( c ) Tyramide signal amplification (TSA) 32 improves biotin detection sensitivity on the yeast surface. In the top row, the three indicated yeast samples (G1 is the winning ligase mutant from the first generation of evolution) were labeled with exogenous biotin for 18 hours then stained for FACS as in (b). The y-axis shows biotinylation extent, and the x-axis quantifies ligase expression level. In the second row, after 18 hours of biotin incubation, yeast were stained with streptavidin-HRP, reacted with biotin-phenol 2 , 32 to create additional biotinylation sites, then stained with streptavidin-phycoerythrin and anti-myc antibody before FACS. The third row omits biotin. Percentage of cells in upper right quadrant (Q2/(Q2+Q4)) shown in top right of each graph. This experiment was performed once, but each yeast sample has been analyzed under identical conditions at least twice in separate experiments with similar results. ( d ) E. coli biotin ligase structure (PDB: 2EWN) with sites mutated in TurboID (left) and miniTurbo (right) shown in red. The N-terminal domain (aa1-63) is also removed from miniTurbo. A non-hydrolyzable analog of biotin-5′-AMP, biotinol-5′-AMP, is shown in yellow stick. ( e ) FACS plots summarizing progress of directed evolution. G1-G3 are the winning clones from generations 1-3 of directed evolution. G3Δ has its N-terminal domain (aa1-63) deleted. Omit biotin samples were grown in biotin-deficient media (see Methods ) for the entire induction period (~18-24 hr). This experiment was performed twice with similar results, except G3Δ omit biotin, which was performed once. ( f ) Comparison of ligase variants in the HEK cytosol showing that TurboID and miniTurbo are much more active than BioID, as well as the starting template and various intermediate clones from the evolution. Indicated ligases were expressed as NES (nuclear export signal) fusions in the HEK cytosol. 50 μM exogenous biotin was added for 3 hours, then whole cell lysates were analyzed by streptavidin blotting. Ligase expression detected by anti-V5 blotting. U, untransfected. S, BirA-R118S. Asterisks indicate ligase self-biotinylation. BioID labeling for 18 hours (50 μM biotin) shown for comparison in the last lane. This experiment was performed twice with similar results. ( g ) Quantitation of streptavidin blot data in (f) and from a 30 minute labeling experiment shown in Supplementary Figure 4b . Quantitation excludes self-biotinylation band. Sum intensity of each lane is divided by the sum intensity of the ligase expression band; ratios are normalized to that of BioID/18 hours, which is set to 1.0. Grey dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity calculated from the two replicates.

    Journal: Nature biotechnology

    Article Title: Efficient proximity labeling in living cells and organisms with TurboID

    doi: 10.1038/nbt.4201

    Figure Lengend Snippet: Directed evolution of TurboID ( a ) Proximity-dependent biotinylation catalyzed by promiscuous biotin ligases. Ligases catalyze the formation of biotin-5′-AMP anhydride, which diffuses out of the active site to biotinylate proximal endogenous proteins on nucleophilic residues such as lysine. ( b ) Yeast display-based selection scheme. A > 10 7 library of ligase variants is displayed on the yeast surface as a fusion to mating protein Aga2p. All ligases have a C-terminal myc epitope tag. Biotin and ATP are added to the yeast library for between 10 minutes and 24 hours. Ligase-catalyzed promiscuous biotinylation is detected by staining with streptavidin-phycoerythrin and ligase expression is detected by staining with anti-myc antibody. Two-dimensional FACS sorting enables enrichment of cells displaying a high ratio of streptavidin to myc staining. ( c ) Tyramide signal amplification (TSA) 32 improves biotin detection sensitivity on the yeast surface. In the top row, the three indicated yeast samples (G1 is the winning ligase mutant from the first generation of evolution) were labeled with exogenous biotin for 18 hours then stained for FACS as in (b). The y-axis shows biotinylation extent, and the x-axis quantifies ligase expression level. In the second row, after 18 hours of biotin incubation, yeast were stained with streptavidin-HRP, reacted with biotin-phenol 2 , 32 to create additional biotinylation sites, then stained with streptavidin-phycoerythrin and anti-myc antibody before FACS. The third row omits biotin. Percentage of cells in upper right quadrant (Q2/(Q2+Q4)) shown in top right of each graph. This experiment was performed once, but each yeast sample has been analyzed under identical conditions at least twice in separate experiments with similar results. ( d ) E. coli biotin ligase structure (PDB: 2EWN) with sites mutated in TurboID (left) and miniTurbo (right) shown in red. The N-terminal domain (aa1-63) is also removed from miniTurbo. A non-hydrolyzable analog of biotin-5′-AMP, biotinol-5′-AMP, is shown in yellow stick. ( e ) FACS plots summarizing progress of directed evolution. G1-G3 are the winning clones from generations 1-3 of directed evolution. G3Δ has its N-terminal domain (aa1-63) deleted. Omit biotin samples were grown in biotin-deficient media (see Methods ) for the entire induction period (~18-24 hr). This experiment was performed twice with similar results, except G3Δ omit biotin, which was performed once. ( f ) Comparison of ligase variants in the HEK cytosol showing that TurboID and miniTurbo are much more active than BioID, as well as the starting template and various intermediate clones from the evolution. Indicated ligases were expressed as NES (nuclear export signal) fusions in the HEK cytosol. 50 μM exogenous biotin was added for 3 hours, then whole cell lysates were analyzed by streptavidin blotting. Ligase expression detected by anti-V5 blotting. U, untransfected. S, BirA-R118S. Asterisks indicate ligase self-biotinylation. BioID labeling for 18 hours (50 μM biotin) shown for comparison in the last lane. This experiment was performed twice with similar results. ( g ) Quantitation of streptavidin blot data in (f) and from a 30 minute labeling experiment shown in Supplementary Figure 4b . Quantitation excludes self-biotinylation band. Sum intensity of each lane is divided by the sum intensity of the ligase expression band; ratios are normalized to that of BioID/18 hours, which is set to 1.0. Grey dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity calculated from the two replicates.

    Article Snippet: To detect biotinylated proteins, blots were incubated with 0.3 μg/mL streptavidin-HRP (Thermo Fisher S911) in PBST-BSA for 1 hour at room temperature.

    Techniques: Selection, Staining, Expressing, FACS, Amplification, Mutagenesis, Labeling, Incubation, Clone Assay, Quantitation Assay

    TurboID and miniTurbo in flies, worms, and other species ( a ) Comparison of ligases in yeast. EBY100 S. cerevisiae expressing BioID, TurboID, or miniTurbo in the cytosol were treated with 50 μM biotin for 18 hours. Whole cell lysates were blotted with streptavidin-HRP to visualize biotinylated proteins, and anti-V5 antibody to visualize ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation. Bands in untransfected lane are endogenous naturally-biotinylated proteins. This experiment was performed twice with similar results. ( b ) Comparison of ligases in E. coli. Ligases, fused at their N-terminal ends to His6-maltose binding protein, were expressed in the cytosol of BL21 E. coli and 50 μM exogenous biotin was added for 18 hours. Whole cell lysates were analyzed as in (a). This experiment was performed twice with similar results. ( c ) – ( g ) Comparison of ligases in flies. ( c ) Scheme for tissue-specific expression of ligases in the wing disc of D. melanogaster . ptc-Gal4 induces ligase expression in a strip of cells within the wing imaginal disc that borders the anterior/posterior compartments. ( d ) Imaging of larval wing discs after 5 days of growth on biotin-containing food. Biotinylated proteins are detected by staining with streptavidin-AlexaFluor555, and ligase expression is detected by anti-V5 staining. Panels show the pouch region of the wing disc, indicated by the dashed line in (c). Scale bar, 40 μm. Each experimental condition has at least three technical replicates; one representative image is shown. This experiment was independently repeated two times with similar results. ( e ) Quantitation of streptavidin-AlexaFluor555 signal intensities in (d). Error bars, s.e.m. Average fold-change shown as text above bars. Sample size values (n) from left column to right: 5, 6, 3. ( f ) Scheme for ubiquitous expression of ligases in flies, at all developmental timepoints, via the act-Gal4 driver. ( g ) Western blotting of fly lysates prepared as in (f). Biotinylated proteins detected by blotting with streptavidin-HRP, ligase expression detected by anti-V5 blotting. In control sample, act-Gal4 drives expression of UAS-luciferase. Bands in control lanes are endogenous naturally-biotinylated proteins. This experiment was performed twice with similar results. ( h ) – ( k ) Comparison of ligases in worms. ( h ) Scheme for tissue-specific expression of ligases in C. elegans intestine via ges-1p promoter. Transgenic strains are fed either biotin-producing E. coli OP50 (biotin+), or biotin-auxotrophic E. coli MG1655bioB:kan (biotin-). Promoter ges-1p drives ligase expression approximately 150 minutes after the first cell cleavage. ( i ) Adult worms prepared as in (h) were shifted to 25°C for one generation, then lysed and analyzed by Western blotting. Control worms (N2) do not express ligase. Anti-HA antibody detects ligase expression. Streptavidin-IRDye detects biotinylated proteins. This experiment was performed five times (n = 5). In biotin+ conditions, BioID biotinylation activity was undetectable and TurboID gave robust biotinylation signal (n = 5/5). Despite high activity detected by immunofluorescence in embryos, we only detected some low level of biotinylation by miniTurbo in adults (n = 2/5), likely due to its low expression levels. ( j ) Representative images of bean stage worm embryos (stage 1) from (h). See Supplementary Figure 15a for representative images of comma stage worm embryos (stage 2). Embryos were fixed and stained with streptavidin-AF488 to detect biotinylated proteins, and anti-HA antibody to detect ligase expression. Intestine is outlined by a white dotted line. Scale bar, 10 μm. Quantitation of multiple replicates shown in (k). ( k ) Quantitation of streptavidin-AF488 signal acquired from IF staining of embryonic stages 1 and 2 shown in (j) and Supplementary Figure 15a . Mean streptavidin pixel intensities for each embryo assessed are plotted for BioID (B), TurboID (T), and miniTurbo (mT). Two independent transgenic lines for BioID and TurboID and one for miniTurbo were assessed. Number of embryos imaged (n) from left to right: 26, 18, 11, 16, 25, 8, 19, 23, 14, 14, 23, 9. Statistical significance via Mann-Whitney U test (two-sided). ***p ≤ 0.0001, **p ≤ 0.001, *p ≤ 0.01. Pink asterisks indicate significance of pairwise comparisons between biotin- and corresponding biotin+ treated embryos. Mean (reported in Supplementary Figure 15b ) is shown as a black horizontal line for each condition, and error bars indicate s.e.m. Note that the streptavidin-AF488 pixel intensities for miniTurbo are an underrepresentation of the signal as camera exposure settings were lowered to avoid pixel saturation (see Methods ). See Supplementary Figure 15 for more details.

    Journal: Nature biotechnology

    Article Title: Efficient proximity labeling in living cells and organisms with TurboID

    doi: 10.1038/nbt.4201

    Figure Lengend Snippet: TurboID and miniTurbo in flies, worms, and other species ( a ) Comparison of ligases in yeast. EBY100 S. cerevisiae expressing BioID, TurboID, or miniTurbo in the cytosol were treated with 50 μM biotin for 18 hours. Whole cell lysates were blotted with streptavidin-HRP to visualize biotinylated proteins, and anti-V5 antibody to visualize ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation. Bands in untransfected lane are endogenous naturally-biotinylated proteins. This experiment was performed twice with similar results. ( b ) Comparison of ligases in E. coli. Ligases, fused at their N-terminal ends to His6-maltose binding protein, were expressed in the cytosol of BL21 E. coli and 50 μM exogenous biotin was added for 18 hours. Whole cell lysates were analyzed as in (a). This experiment was performed twice with similar results. ( c ) – ( g ) Comparison of ligases in flies. ( c ) Scheme for tissue-specific expression of ligases in the wing disc of D. melanogaster . ptc-Gal4 induces ligase expression in a strip of cells within the wing imaginal disc that borders the anterior/posterior compartments. ( d ) Imaging of larval wing discs after 5 days of growth on biotin-containing food. Biotinylated proteins are detected by staining with streptavidin-AlexaFluor555, and ligase expression is detected by anti-V5 staining. Panels show the pouch region of the wing disc, indicated by the dashed line in (c). Scale bar, 40 μm. Each experimental condition has at least three technical replicates; one representative image is shown. This experiment was independently repeated two times with similar results. ( e ) Quantitation of streptavidin-AlexaFluor555 signal intensities in (d). Error bars, s.e.m. Average fold-change shown as text above bars. Sample size values (n) from left column to right: 5, 6, 3. ( f ) Scheme for ubiquitous expression of ligases in flies, at all developmental timepoints, via the act-Gal4 driver. ( g ) Western blotting of fly lysates prepared as in (f). Biotinylated proteins detected by blotting with streptavidin-HRP, ligase expression detected by anti-V5 blotting. In control sample, act-Gal4 drives expression of UAS-luciferase. Bands in control lanes are endogenous naturally-biotinylated proteins. This experiment was performed twice with similar results. ( h ) – ( k ) Comparison of ligases in worms. ( h ) Scheme for tissue-specific expression of ligases in C. elegans intestine via ges-1p promoter. Transgenic strains are fed either biotin-producing E. coli OP50 (biotin+), or biotin-auxotrophic E. coli MG1655bioB:kan (biotin-). Promoter ges-1p drives ligase expression approximately 150 minutes after the first cell cleavage. ( i ) Adult worms prepared as in (h) were shifted to 25°C for one generation, then lysed and analyzed by Western blotting. Control worms (N2) do not express ligase. Anti-HA antibody detects ligase expression. Streptavidin-IRDye detects biotinylated proteins. This experiment was performed five times (n = 5). In biotin+ conditions, BioID biotinylation activity was undetectable and TurboID gave robust biotinylation signal (n = 5/5). Despite high activity detected by immunofluorescence in embryos, we only detected some low level of biotinylation by miniTurbo in adults (n = 2/5), likely due to its low expression levels. ( j ) Representative images of bean stage worm embryos (stage 1) from (h). See Supplementary Figure 15a for representative images of comma stage worm embryos (stage 2). Embryos were fixed and stained with streptavidin-AF488 to detect biotinylated proteins, and anti-HA antibody to detect ligase expression. Intestine is outlined by a white dotted line. Scale bar, 10 μm. Quantitation of multiple replicates shown in (k). ( k ) Quantitation of streptavidin-AF488 signal acquired from IF staining of embryonic stages 1 and 2 shown in (j) and Supplementary Figure 15a . Mean streptavidin pixel intensities for each embryo assessed are plotted for BioID (B), TurboID (T), and miniTurbo (mT). Two independent transgenic lines for BioID and TurboID and one for miniTurbo were assessed. Number of embryos imaged (n) from left to right: 26, 18, 11, 16, 25, 8, 19, 23, 14, 14, 23, 9. Statistical significance via Mann-Whitney U test (two-sided). ***p ≤ 0.0001, **p ≤ 0.001, *p ≤ 0.01. Pink asterisks indicate significance of pairwise comparisons between biotin- and corresponding biotin+ treated embryos. Mean (reported in Supplementary Figure 15b ) is shown as a black horizontal line for each condition, and error bars indicate s.e.m. Note that the streptavidin-AF488 pixel intensities for miniTurbo are an underrepresentation of the signal as camera exposure settings were lowered to avoid pixel saturation (see Methods ). See Supplementary Figure 15 for more details.

    Article Snippet: To detect biotinylated proteins, blots were incubated with 0.3 μg/mL streptavidin-HRP (Thermo Fisher S911) in PBST-BSA for 1 hour at room temperature.

    Techniques: Expressing, Binding Assay, Stripping Membranes, Imaging, Staining, Quantitation Assay, Activated Clotting Time Assay, Western Blot, Luciferase, Transgenic Assay, Activity Assay, Immunofluorescence, MANN-WHITNEY

    Comparison of histone extraction protocols, and specificity testing of streptavidin and anti-biotin Panel A: Nuclear histones were extracted from Jurkat cells (lanes 1, 5, and 9) or HeLa cells (lanes 3, 7, and 11) by using HCl; for comparison histones were extracted from Jurkat cells (lanes 2, 6, and 10) or HeLa cells (lanes 4, 8, and 12) by using H2 SO4 +TCA+acetone/HCl+acetone. Histones were probed with coomassie blue (lanes 1–4) and antibodies to the C-termini in histone H3 (lanes 5–8) and H4 (lanes 9–12). Panel B: HCl extracts of Jurkat cell histones (lanes 1, 4, 7, 10, and 13), recombinant human histone H4 (lanes 2, 5, 8, 11, and 14), and chemically biotinylated histone H4 (lanes 3, 6, 9, 12, and 15) were probed with streptavidin without biotin competitor (lanes 1–3) and with 5 mM free biotin (lanes 4–6), and with anti-biotin without biotin competitor (lanes 7–9) and with 5 mM free biotin (lanes 10–12), and with coomassie blue (lanes 13–15).

    Journal:

    Article Title: Biotinylation is a natural, albeit rare, modification of human histones

    doi: 10.1016/j.ymgme.2011.08.030

    Figure Lengend Snippet: Comparison of histone extraction protocols, and specificity testing of streptavidin and anti-biotin Panel A: Nuclear histones were extracted from Jurkat cells (lanes 1, 5, and 9) or HeLa cells (lanes 3, 7, and 11) by using HCl; for comparison histones were extracted from Jurkat cells (lanes 2, 6, and 10) or HeLa cells (lanes 4, 8, and 12) by using H2 SO4 +TCA+acetone/HCl+acetone. Histones were probed with coomassie blue (lanes 1–4) and antibodies to the C-termini in histone H3 (lanes 5–8) and H4 (lanes 9–12). Panel B: HCl extracts of Jurkat cell histones (lanes 1, 4, 7, 10, and 13), recombinant human histone H4 (lanes 2, 5, 8, 11, and 14), and chemically biotinylated histone H4 (lanes 3, 6, 9, 12, and 15) were probed with streptavidin without biotin competitor (lanes 1–3) and with 5 mM free biotin (lanes 4–6), and with anti-biotin without biotin competitor (lanes 7–9) and with 5 mM free biotin (lanes 10–12), and with coomassie blue (lanes 13–15).

    Article Snippet: Controls included pre-immune serum, horseradish peroxidase-conjugated streptavidin (Thermo Scientific), and IRDye 800CW Streptavidin (LI-COR).

    Techniques: Recombinant

    Biotinylation marks can be detected in bulk extracts from human cells, using streptavidin and anti-biotin as probes Bulk extracts of histone extracts from various cell lineages were probed with streptavidin (panel A), anti-biotin (panel B), and the loading and transfer controls coomassie blue (panel C), anti-H3 (panel D), and anti-H4 (panel E). Panel F: Histones from HeLa cells were extracted with H2 SO4 +TCA+acetone/HCl+acetone. Ten or five microgram of histones were loaded per well. Blots were blocked with PBS containing 5% BSA. After probing with horseradish peroxidase-conjugated anti-biotin or Nutravidin, the blots were washed for 6 hrs, and exposed to autoradiography film for 1 min.

    Journal:

    Article Title: Biotinylation is a natural, albeit rare, modification of human histones

    doi: 10.1016/j.ymgme.2011.08.030

    Figure Lengend Snippet: Biotinylation marks can be detected in bulk extracts from human cells, using streptavidin and anti-biotin as probes Bulk extracts of histone extracts from various cell lineages were probed with streptavidin (panel A), anti-biotin (panel B), and the loading and transfer controls coomassie blue (panel C), anti-H3 (panel D), and anti-H4 (panel E). Panel F: Histones from HeLa cells were extracted with H2 SO4 +TCA+acetone/HCl+acetone. Ten or five microgram of histones were loaded per well. Blots were blocked with PBS containing 5% BSA. After probing with horseradish peroxidase-conjugated anti-biotin or Nutravidin, the blots were washed for 6 hrs, and exposed to autoradiography film for 1 min.

    Article Snippet: Controls included pre-immune serum, horseradish peroxidase-conjugated streptavidin (Thermo Scientific), and IRDye 800CW Streptavidin (LI-COR).

    Techniques: Autoradiography