Horseradish peroxidase hrp conjugated anti mouse or anti rabbit igg antibody

K562 cells were collected before induction of differentiation, as well as three days after treatment with PMA or hemin, washed once with PBS, and frozen at −80°C. Cell lysates were prepared as previously described [12 (link)]. Lysates were diluted in 3X protein loading buffer (187.5 mM Tris (pH 6.8), 30% glycerol, 6% SDS, 0.005% pyronin Y, 5% beta-mercaptoethanol) and boiled for 5 minutes at 95°C. Lysate proteins were separated by gel electrophoresis using Mini-PROTEAN TGX Precast Gels (BioRad), and transferred onto nitrocellulose membranes (BioRad). The membranes were blocked for 1 h at RT with 5% dried milk or 5% BSA in TBST buffer (Tris-buffered saline + 0.1% Tween 20). Membranes were incubated with primary antibody in TBST + 5% milk or BSA with gentle shaking for 2 h at room temperature or overnight at 4 °C. Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse- or anti-rabbit-IgG antibody (Cell Signaling). Protein was detected using Chemiluminescent HRP Substrate (Millipore) and film exposure. The SIRT1 (sc-74504), HSC70 (sc-7298), and SIRT7 (sc-135055) antibodies were obtained from Santa Cruz; SIRT2 (#04 1124) and SIRT5 (#ABE198) from Millipore; SIRT3 (#5490) and SIRT6 (#2590) from Cell Signaling.