Mouse monoclonal antibody against human β actin

Total protein from MCF-7 cells was extracted using a cracking buffer [100 mmol/l Tris (pH 6.7), 2% glycerol] containing a protease inhibitor (Sigma-Aldrich) at a 1:200 dilution, resolved on 10% SDS-PAGE for immunoblot analysis and then incubated using custom-made rabbit polyclonal antibody against human-CHK1/CHK2 (Cell Signalling Technology, Inc., Danvers, MA, USA) at 1:100 dilution in 5% nonfat dry milk overnight at 4°C. A mouse monoclonal antibody against human-β-actin (Sigma-Aldrich) at 1:10,000 was used as control. Appropriate horseradish peroxidase-conjugated secondary antibody, either anti-mouse or anti-rabbit (GE Healthcare Life Sciences, Chalfont, UK), was used at 1:2,500 dilution in milk. Immunoblots were developed using the Super Signal West Pico chemiluminescent substrate kit (Pierce Biotechnology, Inc., Rockford, IL, USA) and images were captured using a Digimax i50 digital camera (Samsung, Suwon, South Korea). The density of immunoblot bands was analyzed using Band Leader software (version 3.0; Band Leader Systems, Inc., Boulder City, NV, USA) as described previously (18 (link)).