Phospho mapk family antibody sampler kit 9910

HEK293 cells growing on culture dishes were washed twice with PBS, and then lysed in RIPA lysis buffer (50 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride) for 30 min at 4°C. The samples were then centrifuged (16000 × g for 15 min) to remove residual cell debris, and the supernatant was collected as total protein lysates. Protein concentrations were measured by BCA assay. Protein samples were mixed with 5× Laemmli buffer, incubated at 37°C for 20 min, separated on an 8% SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked in nonfat milk, incubated with primary antibody (anti-myc, 1:200, Santa Cruz, sc-40; anti-BK, 1:200, Alomone Labs, APC-021; anti-MAPK antibodies, 1:2000, Cell Signaling Technology, Phospho-MAPK Family Antibody Sampler Kit #9910 and MAPK Family Antibody Sampler Kit #9926) overnight at 4°C and with horseradish-peroxidase-conjugated secondary antibody for 2 h at room temperature. Immunoreactivity was detected with enhanced chemiluminescence (ECL; Pierce, USA) using Alpha Imager Detection System (AlphaInnotech, USA). The intensity of bands was quantified by ImageJ software (NIH, USA).