Review for Mouse Anti-ß-Actin Antibody

Protein extract from MEFs was prepared in radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology) supplied with PMSF, protease inhibitor mixture, sodium orthovanadate, or RNase inhibitor. Lysates were quantified using a Bio-Rad protein assay kit. Forty micrograms of proteins were subjected to SDS-PAGE and transferred onto nitrocellulose membranes and blocked with 5% non-fat milk in TBS-T (20 mMTris, 500 mMNaCl, and 0.1% Tween 20) at room temperature for 1 h with rocking. The membranes were probed with primary Antibody (1/1000 dilution) overnight at 4°C. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary Antibodies (Thermo Scientific, Rockford, IL) in 5% non-fat milk/TBS-T at room temperature for 1 h. The protein–Antibody complex was detected by ECL substrate (cat # 32106; Thermo Scientific).