Clarity or clarity max substrate, by Bio-Rad - Product details

1

Immunoprecipitation and Western Blot Analysis

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HEK-293T cells were harvested 24h post-transfection. For the production of whole-cell lysates, cells were lysed in 300 μl of lysis buffer (50 mM Tris at pH 7.4, 1% NP-40 [v/v], 150 mM NaCl , 1mM GTP, 10mM MgCl2, a protease inhibitor cocktail [1x EDTA-free protease inhibitors, Sigma], 10mM NaF, 1mM Na3VO4, 2μM Bortezomib) at 4°C. 60 μl of lysates was saved as input and the rest was subject to immunoprecipitation.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.

Markiewicz Ł., Uśpieński T., Baran B., Niedziółka S.M, & Niewiadomski P. (2021). Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus. Cellular signalling, 80.

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2

Immunoprecipitation and Western Blot Analysis

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HEK-293T cells were harvested 24h post-transfection. For the production of whole-cell lysates, cells were lysed in 300 μl of lysis buffer (50 mM Tris at pH 7.4, 1% NP-40 [v/v], 150 mM NaCl , 1mM GTP, 10mM MgCl2, a protease inhibitor cocktail [1x EDTA-free protease inhibitors, Sigma], 10mM NaF, 1mM Na3VO4, 2μM Bortezomib) at 4°C. 60 μl of lysates was saved as input and the rest was subject to immunoprecipitation.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.

Markiewicz Ł., Uśpieński T., Niedziółka S.M., & Niewiadomski P. (2020). Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus.

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3

Immunoprecipitation of NCOA7 and E2-crimson

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U87MG cells were stably transduced with vectors carrying RRL.sin.cPPT.CMV/Flag-NCOA7.WPRE or RRL.sin.cPPT.CMV/Flag-E2-crimson-.WPRE and selected with 1 μg/ml puromycin. 15-22 x 10⁶ cells were washed twice in cold PBS and resuspended in lysis buffer (20mM Hepes-NaOH pH 7.5, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail), the lysates clarified by centrifugation at 1000g, for 10 min at 4°C and then incubated with Flag-magnetic beads (Life Technologies) for 3 hr at 4°C. The beads were washed 5 times and the immunoprecipitated proteins eluted using 3x Flag peptide (150 μg/ml, Sigma-Aldrich) for 1.5-2 hr. Cell lysates and immunoprecipitation eluates were supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analyzed by immunoblotting using primary antibodies specific for the Flag epitope (Sigma-Aldrich F3165), tubulin (mouse monoclonal DM1A, Sigma-Aldrich T9026), ATP6V1B2 (Proteintech 15097-1-AP), ATP6V1A (Proteintech 17115-1-AP), ATP6V1E1 (Abcam Ab111733), ATP6V1G2 (Proteintech 25316-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse, or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad). A Bio-Rad ChemiDoc imager was used.

Doyle T., Moncorgé O., Bonaventure B., Pollpeter D., Lussignol M., Tauziet M., Apolonia L., Catanese M.T., Goujon C, & Malim M.H. (2018). The interferon inducible isoform of NCOA7 inhibits endosome-mediated viral entry. Nature microbiology, 3(12), 1369-1376.

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4

SARS-CoV-2 Protein Expression Analysis

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Cells were lysed in lysis buffer (10 mM Tris [pH 7.6], NaCl 150 mM, Triton X-100 1%, EDTA 1 mM, deoxycholate 0.1%) supplemented with sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 5% glycerol, 100 mM dithiothreitol [DTT], 0.02% bromphenol blue), resolved by SDS-PAGE and analyzed by immunoblotting using primary antibodies against SARS-CoV nucleocapsid (Bio-Techne NB100-56683), SARS-CoV spike (GeneTex GTX632604), actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (Thermo Fisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad). A Bio-Rad ChemiDoc imager was used.

Rebendenne A., Chaves Valadão A.L., Tauziet M., Maarifi G., Bonaventure B., McKellar J., Planès R., Nisole S., Arnaud-Arnould M., Moncorgé O, & Goujon C. (2021). SARS-CoV-2 Triggers an MDA-5-Dependent Interferon Response Which Is Unable To Control Replication in Lung Epithelial Cells. Journal of Virology, 95(8), e02415-20.

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5

Quantitative Immunoblot Analysis of ACE2

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Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromophenol blue), resolved by SDS-PAGE and analyzed by immunoblotting using primary antibodies against ACE2 (ProteinTech 21115–1-P) and Actin (Sigma-Aldrich A1978), followed by HRP-conjugated anti-rabbit or anti-mouse immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad). A Bio-Rad ChemiDoc imager was used.

Rebendenne A., Roy P., Bonaventure B., Chaves V.A., Desmarets L., Rouillé Y., Tauziet M., Arnaud-Arnould M., Giovannini D., Lee Y., DeWeirdt P., Hegde M., Garcia de G.F., McKellar J., Wencker M., Dubuisson J., Belouzard S., Moncorgé O., Doench J.G, & Goujon C. (2021). Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs. Research Square.

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6

Immunoprecipitation of NCOA7 and E2-crimson

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U87MG cells were stably transduced with vectors carrying RRL.sin.cPPT.CMV/Flag-NCOA7.WPRE or RRL.sin.cPPT.CMV/Flag-E2-crimson-.WPRE and selected with 1 μg/ml puromycin. 15-22 x 10⁶ cells were washed twice in cold PBS and resuspended in lysis buffer (20mM Hepes-NaOH pH 7.5, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail), the lysates clarified by centrifugation at 1000g, for 10 min at 4°C and then incubated with Flag-magnetic beads (Life Technologies) for 3 hr at 4°C. The beads were washed 5 times and the immunoprecipitated proteins eluted using 3x Flag peptide (150 μg/ml, Sigma-Aldrich) for 1.5-2 hr. Cell lysates and immunoprecipitation eluates were supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analyzed by immunoblotting using primary antibodies specific for the Flag epitope (Sigma-Aldrich F3165), tubulin (mouse monoclonal DM1A, Sigma-Aldrich T9026), ATP6V1B2 (Proteintech 15097-1-AP), ATP6V1A (Proteintech 17115-1-AP), ATP6V1E1 (Abcam Ab111733), ATP6V1G2 (Proteintech 25316-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse, or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad). A Bio-Rad ChemiDoc imager was used.

Doyle T., Moncorgé O., Bonaventure B., Pollpeter D., Lussignol M., Tauziet M., Apolonia L., Catanese M.T., Goujon C, & Malim M.H. (2018). The interferon inducible isoform of NCOA7 inhibits endosome-mediated viral entry. Nature microbiology, 3(12), 1369-1376.

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Clarity or clarity max substrate

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6 protocols using clarity or clarity max substrate

Immunoprecipitation and Western Blot Analysis

Immunoprecipitation and Western Blot Analysis

Immunoprecipitation of NCOA7 and E2-crimson

SARS-CoV-2 Protein Expression Analysis

Quantitative Immunoblot Analysis of ACE2

Immunoprecipitation of NCOA7 and E2-crimson

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