Ecl supersignal west pico plus chemiluminescent substrate

Cells were pelleted by centrifugation (1,500 rpm, 5 min, at 4 °C) and washed in cold phosphate buffered saline (PBS). Cell pellets were lysed in RIPA Lysis and Extraction Buffer. Mouse testis lysate was prepared by homogenization in T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific; 78510). Tissue and cell debris were removed by centrifugation (13,000 rpm, 10 min, at 4 °C). Protein concentrations were determined by using Pierce BCA Protein Assay Kits (Pierce; Cat: #23225). For Western blot analysis of hBRCA1 and mBRCA1 proteins, 20-30 μg of each lysate was separated by 6% Novex Tris-Glycine gel (Invitrogen). Membranes were probed with anti-BRCA1 antibodies overnight at 4 °C with gentle shaking. And the corresponding horseradish peroxidase (HRP) conjugated secondary antibodies were used. Proteins were visualized using ECL SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, Cat. #34580).