Hrp conjugated trueblot secondary antibodies

Total cell lysates were prepared by lysing the cells in RIPA buffer (1% NP40; 50 mM Tris, pH 7.5; 150 mM NaCl) supplemented with a protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and 1 mM sodium orthovanadate. Immunoprecipitations were performed using GFP conjugated sepharose beads (Allele Biotechnology, San Diego, CA, USA), mouse monoclonal HA beads (Sigma-Aldrich) and acetyl-lysine conjugated agarose beads (Immunechem, BC, Canada). Anti-GFP antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-HA antibody from Covance and anti-RFP antibody from Evrogen (Moscow, Russia). HRP conjugated Trueblot secondary antibodies were purchased from eBioscience (eBioscience Inc., San Diego, CA, USA) and Western blots were developed using West Pico or West Femto enhanced ECL detection system (Thermo Scientific, Rockford, IL, USA).

Total cell lysates were prepared by lysing the cells in modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP-40) supplemented with 100 μg/ml leupeptin, 100 μg/ml aprotinin and 1 mM sodium orthovanadate. BRCA1 and ALDH1 antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA., Actin antibodies were from Sigma (St. Louis MO) and EpCAM antibodies were from AbCam. HRP conjugated Trueblot secondary antibodies were purchased from eBioscience (eBioscience Inc. San Diego, CA) and western blots were developed using a Pierce ECL detection system (Thermo Scientific, Rockford IL).