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.
Ab111733
Manufactured by Proteintech
Ab111733 is a primary antibody produced by Proteintech. It is designed for research use only.
Automatically generated - may contain errors
2 protocols using ab111733
1
Immunoprecipitation of NCOA7 and E2-crimson
2
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.
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