Amersham ecl select western blotting detection kit

Cells were collected by trypsinization and washed with phosphate buffered saline (PBS). Samples were then incubated in lysis buffer [50 mM Tris, 0.5% NP40, 1 mM EDTA, 10% glycerol, 400 mM sodium chloride and Protease Inhibitor Cocktail (Roche)] and cleared by centrifuging at 20 000 × g, 4°C for 30 min. Protein concentration was determined with BCA Protein Assay Reagent (Thermo Scientific). From each sample, 10 μg protein were resolved by SDS/PAGE and subsequently transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with 5% non-fat dry milk in PBST buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.46 mM KH₂PO₄, 0.05% Tween-20) for 1 h at room temperature and incubated with Oct3/4 or GAPDH antibodies (Santa Cruz) overnight. Membrane were then washed three times with PBST buffer and incubated with rabbit anti-goat-HRP or goat anti-mouse-HRP (GE technology) at a dilution of 1:20 000 in PBST for 1 h at room temperature. Signals were detected using the Amersham ECL Select Western Blotting Detection Kit (GE Health Care Life Sciences) and exposed to Super RX-N film (Fuji).