horseradish peroxidase-conjugated streptavidin Search Results


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  • 95
    Thermo Fisher streptavidin horseradish peroxidase
    <t>Streptavidin</t> DNA binding assay with mutant p53 binding to unique gene sequences
    Streptavidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore streptavidin horseradish peroxidase conjugate
    Missorted surface proteins accumulate in the sortase mutant AR01. A. Wild type S . pyogenes D471, sortase mutant AR01, complemented AR01+pAR107, and a passaged AR01 variant, were diluted from overnight cultures 1:100 into fresh media (containing spectinomycin for AR01+pAR107), grown to log phase, fixed, attached to glass cover slides, and permeabilized with PlyC. The cells were blocked and incubated with pentaglycine-biotin (5G-biotin) in the presence or absence of purified S . aureus sortase A. The slides were washed, and pentaglycine-biotins molecules (attached to LPXTG motifs by sortase) were detected using <t>FITC-streptavidin.</t> DAPI was used to visualize DNA. The scale bar represents 1 μm. Note that expression of sortase from pAR107 is lower than the wild type, explaining the presence of intact LPXTG motifs. B. The cell wall fraction of log-phase D471 cells (solubilized with PlyC in PBS 30% raffinose) was separated by SDS-PAGE. Duplicate gels were stained with GelCode blue or processed for Western blot using the 10B6 monoclonal antibody. The most prominent bands on the stained gel correspond to M protein.
    Streptavidin Horseradish Peroxidase Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher horseradish peroxidase hrp conjugated streptavidin
    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with <t>HRP-conjugated</t> <t>streptavidin</t> followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.
    Horseradish Peroxidase Hrp Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher streptavidin hrp
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3879 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biogenex horseradish peroxidase conjugated streptavidin
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Biogenex, used in various techniques. Bioz Stars score: 92/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson horseradish peroxidase conjugated streptavidin
    The specificity of IGF signaling antibody array. After incubated with fluorescence conjugated <t>streptavidin,</t> the array was scanned by Genpix 4000B and the specific signal of each subarray was visualized. The signal intensity was extracted by Genpix software, and showed the array specificity as well.
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novocastra horseradish peroxidase conjugated streptavidin
    The specificity of IGF signaling antibody array. After incubated with fluorescence conjugated <t>streptavidin,</t> the array was scanned by Genpix 4000B and the specific signal of each subarray was visualized. The signal intensity was extracted by Genpix software, and showed the array specificity as well.
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Novocastra, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: <t>streptavidin</t> labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 825 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies horseradish peroxidase conjugated streptavidin
    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: <t>streptavidin</t> labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 432 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boster Bio horseradish peroxidase conjugated streptavidin
    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: <t>streptavidin</t> labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Boster Bio, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare horseradish peroxidase conjugated streptavidin
    CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by <t>streptavidin-HRP</t> (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Nichirei horseradish peroxidase conjugated streptavidin
    CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by <t>streptavidin-HRP</t> (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Nichirei, used in various techniques. Bioz Stars score: 93/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Ventana Medical horseradish peroxidase conjugated streptavidin
    CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by <t>streptavidin-HRP</t> (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad horseradish peroxidase conjugated streptavidin
    CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by <t>streptavidin-HRP</t> (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SouthernBiotech horseradish peroxidase conjugated streptavidin
    CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by <t>streptavidin-HRP</t> (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega horseradish peroxidase conjugated streptavidin
    CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by <t>streptavidin-HRP</t> (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Biogenex horseradish peroxidase streptavidin conjugate
    CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by <t>streptavidin-HRP</t> (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).
    Horseradish Peroxidase Streptavidin Conjugate, supplied by Biogenex, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Trinity Biotech horseradish peroxidase streptavidin conjugate
    Standard curves of calretinin enzyme-linked immunosorbent assay (ELISA) systems . Assay variant 1: Capture and detection antibodies based on Schierle et al. [ 18 ]. Assay variant 2: Modification of variant 1 with inversion of capture and detection antibodies and amplification by a <t>biotin-streptavidin</t> complex. Assay variant 3: Sandwich ELISA with novel polyclonal anti-calretinin rabbit IgG antibody for capturing and detection (biotinylated).
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    Thermo Fisher streptavidin hrp conjugate
    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
    Streptavidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 857 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase conjugate
    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on <t>streptavidin</t> magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.
    Streptavidin Horseradish Peroxidase Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1012 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche horseradish peroxidase conjugated streptavidin
    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on <t>streptavidin</t> magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase hrp conjugate
    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on <t>streptavidin</t> magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.
    Streptavidin Horseradish Peroxidase Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime horseradish peroxidase conjugated streptavidin
    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on <t>streptavidin</t> magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Streptavidin DNA binding assay with mutant p53 binding to unique gene sequences

    Journal:

    Article Title: Three assays show differences in binding of wild-type and mutant p53 to unique gene sequences

    doi:

    Figure Lengend Snippet: Streptavidin DNA binding assay with mutant p53 binding to unique gene sequences

    Article Snippet: This membrane was incubated after blocking in a solution containing 1:100,000 dilution of streptavidin horseradish peroxidase (Pierce, Rockford, IL) and visualized using a chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce).

    Techniques: DNA Binding Assay, Mutagenesis, Binding Assay

    Streptavidin DNA binding assay with wild-type p53 binding to unique gene sequences

    Journal:

    Article Title: Three assays show differences in binding of wild-type and mutant p53 to unique gene sequences

    doi:

    Figure Lengend Snippet: Streptavidin DNA binding assay with wild-type p53 binding to unique gene sequences

    Article Snippet: This membrane was incubated after blocking in a solution containing 1:100,000 dilution of streptavidin horseradish peroxidase (Pierce, Rockford, IL) and visualized using a chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce).

    Techniques: DNA Binding Assay, Binding Assay

    Competition experiment with streptavidin DNA binding assay

    Journal:

    Article Title: Three assays show differences in binding of wild-type and mutant p53 to unique gene sequences

    doi:

    Figure Lengend Snippet: Competition experiment with streptavidin DNA binding assay

    Article Snippet: This membrane was incubated after blocking in a solution containing 1:100,000 dilution of streptavidin horseradish peroxidase (Pierce, Rockford, IL) and visualized using a chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce).

    Techniques: DNA Binding Assay

    Missorted surface proteins accumulate in the sortase mutant AR01. A. Wild type S . pyogenes D471, sortase mutant AR01, complemented AR01+pAR107, and a passaged AR01 variant, were diluted from overnight cultures 1:100 into fresh media (containing spectinomycin for AR01+pAR107), grown to log phase, fixed, attached to glass cover slides, and permeabilized with PlyC. The cells were blocked and incubated with pentaglycine-biotin (5G-biotin) in the presence or absence of purified S . aureus sortase A. The slides were washed, and pentaglycine-biotins molecules (attached to LPXTG motifs by sortase) were detected using FITC-streptavidin. DAPI was used to visualize DNA. The scale bar represents 1 μm. Note that expression of sortase from pAR107 is lower than the wild type, explaining the presence of intact LPXTG motifs. B. The cell wall fraction of log-phase D471 cells (solubilized with PlyC in PBS 30% raffinose) was separated by SDS-PAGE. Duplicate gels were stained with GelCode blue or processed for Western blot using the 10B6 monoclonal antibody. The most prominent bands on the stained gel correspond to M protein.

    Journal: PLoS ONE

    Article Title: Streptococcus pyogenes Sortase Mutants Are Highly Susceptible to Killing by Host Factors Due to Aberrant Envelope Physiology

    doi: 10.1371/journal.pone.0140784

    Figure Lengend Snippet: Missorted surface proteins accumulate in the sortase mutant AR01. A. Wild type S . pyogenes D471, sortase mutant AR01, complemented AR01+pAR107, and a passaged AR01 variant, were diluted from overnight cultures 1:100 into fresh media (containing spectinomycin for AR01+pAR107), grown to log phase, fixed, attached to glass cover slides, and permeabilized with PlyC. The cells were blocked and incubated with pentaglycine-biotin (5G-biotin) in the presence or absence of purified S . aureus sortase A. The slides were washed, and pentaglycine-biotins molecules (attached to LPXTG motifs by sortase) were detected using FITC-streptavidin. DAPI was used to visualize DNA. The scale bar represents 1 μm. Note that expression of sortase from pAR107 is lower than the wild type, explaining the presence of intact LPXTG motifs. B. The cell wall fraction of log-phase D471 cells (solubilized with PlyC in PBS 30% raffinose) was separated by SDS-PAGE. Duplicate gels were stained with GelCode blue or processed for Western blot using the 10B6 monoclonal antibody. The most prominent bands on the stained gel correspond to M protein.

    Article Snippet: Biotinylated goat anti-rabbit antibody (Sigma) was used at a 1:5000 dilution, and streptavidin horseradish peroxidase conjugate (Sigma) was used at a 1:5000 dilution.

    Techniques: Mutagenesis, Variant Assay, Incubation, Purification, Expressing, SDS Page, Staining, Western Blot

    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with HRP-conjugated streptavidin followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of ErbB-2 Mitogenic and Transforming Activity by RALT, a Mitogen-Induced Signal Transducer Which Binds to the ErbB-2 Kinase Domain †

    doi:

    Figure Lengend Snippet: RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with HRP-conjugated streptavidin followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.

    Article Snippet: RALT proteins captured by the filter were detected by horseradish peroxidase (HRP)-conjugated streptavidin (Pierce) (0.1 μg/ml) followed by enhanced chemiluminescence (ECL).

    Techniques: Incubation, Recombinant, Binding Assay, Mutagenesis, Purification, Affinity Chromatography, SDS Page, Staining, Labeling, Transfection, Expressing, Immunoprecipitation, Western Blot

    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Journal: BMC Cell Biology

    Article Title: A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells

    doi: 10.1186/s12860-014-0045-1

    Figure Lengend Snippet: Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Article Snippet: Each diluted standard was incubated with primary antibody coated on the well and streptavidin-HRP and OD value for each sample was detected by ELISA.

    Techniques: Labeling, Western Blot, Synthesized, Conjugation Assay, Transfection, Purification, Immunoprecipitation, Autoradiography

    The specificity of IGF signaling antibody array. After incubated with fluorescence conjugated streptavidin, the array was scanned by Genpix 4000B and the specific signal of each subarray was visualized. The signal intensity was extracted by Genpix software, and showed the array specificity as well.

    Journal: PLoS ONE

    Article Title: Development of IGF Signaling Antibody Arrays for the Identification of Hepatocellular Carcinoma Biomarkers

    doi: 10.1371/journal.pone.0046851

    Figure Lengend Snippet: The specificity of IGF signaling antibody array. After incubated with fluorescence conjugated streptavidin, the array was scanned by Genpix 4000B and the specific signal of each subarray was visualized. The signal intensity was extracted by Genpix software, and showed the array specificity as well.

    Article Snippet: Horseradish peroxidase-conjugated streptavidin was purchased from BD PharMingen.

    Techniques: Ab Array, Incubation, Fluorescence, Software

    Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: streptavidin labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).

    Journal: The Journal of General Physiology

    Article Title: Mouse Bestrophin-2 Is a Bona fide Cl− Channel

    doi: 10.1085/jgp.200409031

    Figure Lengend Snippet: Localization of mBest2 in the plasma membrane of HEK-293 cells. Lanes 1 and 2: streptavidin labeling of immunoprecipitated proteins. Nontransfected (lane 2) or mBest2-transfected (lane 1) HEK-293 cells were labeled with membrane-impermeant NHS-LC-biotin and lysed. mBest2 was immunoprecipitated with A7116 antibody, run on SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with HRP-conjugated streptavidin. A 64kD band was observed in transfected, but not nontransfected cells. Lanes 3–5: Western blots. Lane 3: total extract from mBest2-transfected cells was probed with antibody to α-actin. Lanes 4 and 5: the extract from mBest2-transfected cells was incubated with streptavidin-beads to collect biotinylated proteins. Biotinylated proteins were then probed with antibody to α-actin (Lane 4) or B4947 mBest2-specific antibody (Lane 5).

    Article Snippet: Depending on the experiment, the nitrocellulose was probed with (a) primary antibody (1/1,000 dilution) followed by horseradish peroxidase–conjugated goat anti–rabbit IgG (1/7,000) (Jackson ImmunoResearch Laboratories) in PBS-T with 1% dry milk or (b) horseradish peroxidase–conjugated streptavidin (Pierce Chemical Co.).

    Techniques: Labeling, Immunoprecipitation, Transfection, SDS Page, Western Blot, Incubation

    CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by streptavidin-HRP (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).

    Journal: The Journal of Experimental Medicine

    Article Title: Selective Activation of the c-Jun NH2-terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4+ T Cells

    doi: 10.1084/jem.20020383

    Figure Lengend Snippet: CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by streptavidin-HRP (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).

    Article Snippet: Blots were developed using horseradish peroxidase–conjugated streptavidin and the ECL Plus (Amersham Biosciences).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Negative Control, SDS Page, Expressing, Stable Transfection, Transfection, Flow Cytometry, Cytometry, Immunoprecipitation, Western Blot

    Standard curves of calretinin enzyme-linked immunosorbent assay (ELISA) systems . Assay variant 1: Capture and detection antibodies based on Schierle et al. [ 18 ]. Assay variant 2: Modification of variant 1 with inversion of capture and detection antibodies and amplification by a biotin-streptavidin complex. Assay variant 3: Sandwich ELISA with novel polyclonal anti-calretinin rabbit IgG antibody for capturing and detection (biotinylated).

    Journal: BMC Cancer

    Article Title: Development of an enzyme-linked immunosorbent assay for the detection of human calretinin in plasma and serum of mesothelioma patients

    doi: 10.1186/1471-2407-10-242

    Figure Lengend Snippet: Standard curves of calretinin enzyme-linked immunosorbent assay (ELISA) systems . Assay variant 1: Capture and detection antibodies based on Schierle et al. [ 18 ]. Assay variant 2: Modification of variant 1 with inversion of capture and detection antibodies and amplification by a biotin-streptavidin complex. Assay variant 3: Sandwich ELISA with novel polyclonal anti-calretinin rabbit IgG antibody for capturing and detection (biotinylated).

    Article Snippet: 60 min after addition of 1:20000 diluted rabbit anti-goat IgG-biotin conjugate (Sigma, Saint Louis, MO, USA) the plates were washed three times and incubated for another 60 min with horseradish-peroxidase-streptavidin conjugate (dilution 1:20000, Fitzgerald Industries International, Concord, MA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Variant Assay, Modification, Amplification, Sandwich ELISA

    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Journal: ACS nano

    Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter

    doi: 10.1021/nn404945r

    Figure Lengend Snippet: GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Article Snippet: Gluc or GlucB labeled EVs in PBS were spotted onto nitrocellulose membranes at 16, 31, 63, 125, 250, 500 and 1,000 ng protein followed by overnight incubation in 5% BSA fraction V (Invitrogen), immunoblotting with streptavidin-HRP conjugate (Pierce) and chemiluminescence detection of biotinylated EVs with SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) on autoradiography films (Denville Scientific, Metuchen, NJ).

    Techniques: Expressing, Live Cell Imaging, Stable Transfection, Transduction, Western Blot, Negative Control, Transmission Assay, Transmission Electron Microscopy, Labeling, Immunolabeling, Dot Blot, Isolation

    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on streptavidin magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.

    Journal: Analytical Chemistry

    Article Title: Robust, Quantitative Analysis of Proteins using Peptide Immunoreagents, in Vitro Translation, and an Ultrasensitive Acoustic Resonant Sensor

    doi: 10.1021/ac500084d

    Figure Lengend Snippet: AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on streptavidin magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.

    Article Snippet: Plates were washed with wash buffer [1× PBS + 0.1% (v/v) Tween-20] and blocked with 1× PBS + 5% (w/v) BSA for 2 h. In each well, 100 μL of sample and 100 μL of 33 nM biotinylated Bcl-xL were added and incubated for 4 h. Plates were washed, incubated with streptavidin horseradish peroxidase conjugate (Strep-HRP, Thermo Scientific) for 1 h, washed, and incubated with TMB substrate (Thermo Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Magnetic Beads, Labeling, Concentration Assay, Competitive Binding Assay