Cell lysis buffer for western or ip

Sperm cell suspensions at a density of 10⁵ cells/mL and the buccal/vaginal epithelial cell suspensions of 10⁴ cells/mL were used for Western blot analysis. Total protein extracts were collected by using cell lysis buffer for western or IP (Beyotime, #P0013, China). The total protein concentration of each sample was measured using an enhanced BCA protein Assay Kit (Beyotime, #P0009, China). Equal amounts of total proteins (60 μg) from each sample were resolved via 10% SDS-PAGE gel and transferred to a nitrocellulose filter membrane 200 mA for 1 hr at ice-water mixture. The blot was blocked with 5% non-fat dry milk in PBST (500 mL 1 × PBS + 1 mL Tween20) for 1 hr at room temperature and then incubated with 1:500 diluted anti-AKAP3 monoclonal antibodies (Abcam, #170856, UK) and 1:2000 diluted anti-GAPDH monoclonal antibodies (Cell Signaling Technology, #5174, USA) separately overnight at 4 °C. On the next day the blots were washed three times with PBST for 10 mins each time followed by incubation with secondary HRP-linked antibody (Cell Signaling Technology, #7074S, 1:2000, USA) for 1 hr at room temperature. Bands were scanned using a densitometer (Bio-Rad, USA).