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  • 99
    Thermo Fisher neutravidin
    Neutravidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/neutravidin/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    neutravidin - by Bioz Stars, 2021-04
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher neutravidin beads
    Validation and high-throughput biomarker assays by Western blotting and ELISA. A , Pull-down and Western blot analysis of SrtA-labeled proteins. MDA-MB-231, Panc-1 and HeLa cells were treated with DMSO or increasing concentration of IMP-1088 NMT inhibitor, lysed and labeled with SrtA overnight; biotinylated proteins were enriched on <t>NeutrAvidin</t> beads, resolved by SDS-PAGE and blotted against YES1 (60 kDa), PRKACA (40 kDa), ARL1 (20 kDa) and GAPDH as loading control (36 kDa). SN: supernatant. NMT substrate proteins show concentration-dependent increase in enrichment with increasing concentration of NMT inhibitor, whereas the low molecular weight of ARL1 also allows for direct identification of the SrtA-labeled fraction in the input sample because of the gel shift induced by ligation to Biotin-ALPET. B , Streptavidin shift analysis of SrtA-labeled proteins. SrtA-labeled samples were briefly incubated with streptavidin before SDS-PAGE and blotting against ARL1 and PRKACA. SrtA biotinylated ARL1 (B-ARL1) shows a molecular weight shift induced by the Biotin-ALPET label that gets shifted by 30 kDa on Streptavidin binding. Biotinylated PRKACA (B-PRKACA) shows no apparent shift in the absence of streptavidin, but a clear shift of 30 kDa upon addition of streptavidin. These shifts match the apparent molecular weight of streptavidin alone, as shown by Ponceau staining. C , SrtA-ELISA analysis of labeled proteins. After protein precipitation to remove excess depsipeptide, 1 μg labeled protein lysate was applied to each well of a streptavidin-coated 96-well plate and incubated for 3 h at RT. After primary (anti-ARL1) and secondary (anti-rabbit HRP) antibody incubations, turnover of QuantaBlu fluorogenic HRP substrate was monitored for 30 min. Slopes were calculated from the first 10 min (linear range) after fitting to a straight line ( Y = Slope · X + Y intercept ) by nonlinear regression (Insert). Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.
    Neutravidin Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/neutravidin beads/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    neutravidin beads - by Bioz Stars, 2021-04
    99/100 stars
      Buy from Supplier

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    Validation and high-throughput biomarker assays by Western blotting and ELISA. A , Pull-down and Western blot analysis of SrtA-labeled proteins. MDA-MB-231, Panc-1 and HeLa cells were treated with DMSO or increasing concentration of IMP-1088 NMT inhibitor, lysed and labeled with SrtA overnight; biotinylated proteins were enriched on NeutrAvidin beads, resolved by SDS-PAGE and blotted against YES1 (60 kDa), PRKACA (40 kDa), ARL1 (20 kDa) and GAPDH as loading control (36 kDa). SN: supernatant. NMT substrate proteins show concentration-dependent increase in enrichment with increasing concentration of NMT inhibitor, whereas the low molecular weight of ARL1 also allows for direct identification of the SrtA-labeled fraction in the input sample because of the gel shift induced by ligation to Biotin-ALPET. B , Streptavidin shift analysis of SrtA-labeled proteins. SrtA-labeled samples were briefly incubated with streptavidin before SDS-PAGE and blotting against ARL1 and PRKACA. SrtA biotinylated ARL1 (B-ARL1) shows a molecular weight shift induced by the Biotin-ALPET label that gets shifted by 30 kDa on Streptavidin binding. Biotinylated PRKACA (B-PRKACA) shows no apparent shift in the absence of streptavidin, but a clear shift of 30 kDa upon addition of streptavidin. These shifts match the apparent molecular weight of streptavidin alone, as shown by Ponceau staining. C , SrtA-ELISA analysis of labeled proteins. After protein precipitation to remove excess depsipeptide, 1 μg labeled protein lysate was applied to each well of a streptavidin-coated 96-well plate and incubated for 3 h at RT. After primary (anti-ARL1) and secondary (anti-rabbit HRP) antibody incubations, turnover of QuantaBlu fluorogenic HRP substrate was monitored for 30 min. Slopes were calculated from the first 10 min (linear range) after fitting to a straight line ( Y = Slope · X + Y intercept ) by nonlinear regression (Insert). Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A *

    doi: 10.1074/mcp.RA118.001043

    Figure Lengend Snippet: Validation and high-throughput biomarker assays by Western blotting and ELISA. A , Pull-down and Western blot analysis of SrtA-labeled proteins. MDA-MB-231, Panc-1 and HeLa cells were treated with DMSO or increasing concentration of IMP-1088 NMT inhibitor, lysed and labeled with SrtA overnight; biotinylated proteins were enriched on NeutrAvidin beads, resolved by SDS-PAGE and blotted against YES1 (60 kDa), PRKACA (40 kDa), ARL1 (20 kDa) and GAPDH as loading control (36 kDa). SN: supernatant. NMT substrate proteins show concentration-dependent increase in enrichment with increasing concentration of NMT inhibitor, whereas the low molecular weight of ARL1 also allows for direct identification of the SrtA-labeled fraction in the input sample because of the gel shift induced by ligation to Biotin-ALPET. B , Streptavidin shift analysis of SrtA-labeled proteins. SrtA-labeled samples were briefly incubated with streptavidin before SDS-PAGE and blotting against ARL1 and PRKACA. SrtA biotinylated ARL1 (B-ARL1) shows a molecular weight shift induced by the Biotin-ALPET label that gets shifted by 30 kDa on Streptavidin binding. Biotinylated PRKACA (B-PRKACA) shows no apparent shift in the absence of streptavidin, but a clear shift of 30 kDa upon addition of streptavidin. These shifts match the apparent molecular weight of streptavidin alone, as shown by Ponceau staining. C , SrtA-ELISA analysis of labeled proteins. After protein precipitation to remove excess depsipeptide, 1 μg labeled protein lysate was applied to each well of a streptavidin-coated 96-well plate and incubated for 3 h at RT. After primary (anti-ARL1) and secondary (anti-rabbit HRP) antibody incubations, turnover of QuantaBlu fluorogenic HRP substrate was monitored for 30 min. Slopes were calculated from the first 10 min (linear range) after fitting to a straight line ( Y = Slope · X + Y intercept ) by nonlinear regression (Insert). Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.

    Article Snippet: Protein-based enrichment and on-bead digestion: 8 μl NeutrAvidin agarose beads and 32 μl agarose blank beads (Pierce® Control Agarose Resin, ThermoScientific) were used for every sample.

    Techniques: High Throughput Screening Assay, Biomarker Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling, Multiple Displacement Amplification, Concentration Assay, SDS Page, Molecular Weight, Electrophoretic Mobility Shift Assay, Ligation, Incubation, Binding Assay, Staining

    SrtA labeling workflows enable multiple detection methods for NMT activity. Protein samples are resuspended in SrtA reaction buffer, here exemplified by cells from tissue culture; however, in principle this approach is equally applicable to tissues exposed to a change in NMT activity. A , Overnight reaction with SrtA and TAMRA modified depsipeptide substrate (TAMRA-ALPET-Haa) enables in-gel fluorescence analysis of the SrtA-labeled protein lysates. Alternatively, samples subjected to SrtA reaction with biotin-modified depsipeptide substrate (Biotin-ALPET-Haa) are enriched and studied by in-gel analysis ( B ) or proteomics approaches ( C and D ), following dimethylation of NeutrAvidin-coated beads in the case of protein-based enrichment.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A *

    doi: 10.1074/mcp.RA118.001043

    Figure Lengend Snippet: SrtA labeling workflows enable multiple detection methods for NMT activity. Protein samples are resuspended in SrtA reaction buffer, here exemplified by cells from tissue culture; however, in principle this approach is equally applicable to tissues exposed to a change in NMT activity. A , Overnight reaction with SrtA and TAMRA modified depsipeptide substrate (TAMRA-ALPET-Haa) enables in-gel fluorescence analysis of the SrtA-labeled protein lysates. Alternatively, samples subjected to SrtA reaction with biotin-modified depsipeptide substrate (Biotin-ALPET-Haa) are enriched and studied by in-gel analysis ( B ) or proteomics approaches ( C and D ), following dimethylation of NeutrAvidin-coated beads in the case of protein-based enrichment.

    Article Snippet: Protein-based enrichment and on-bead digestion: 8 μl NeutrAvidin agarose beads and 32 μl agarose blank beads (Pierce® Control Agarose Resin, ThermoScientific) were used for every sample.

    Techniques: Labeling, Activity Assay, Modification, Fluorescence, Protein Enrichment