Methanol activated pvdf filter membrane

Frozen tissue samples were homogenized in RIPA buffer consisting of 1% protease inhibitor mixture. The mixture was centrifuged at 14 000 g for 15 min at 4°C and the supernatant was obtained. Total proteins were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), and 30 μg of protein per sample was separated onto a denaturing polyacrylamide gel containing SDS, and then transferred to a methanol-activated PVDF filter membrane (Bio-Rad, Hercules, CA, USA). Before immunodetection, membranes were blocked within 5% nonfat dry milk. Primary antibodies, anti-RRBP1 (1:1000; rabbit polyclonal; Abcam, Cambridge, MA, USA), were diluted in the buffer and incubated at 4°C overnight. After subsequent washing with TBST, membranes were incubated with secondary antibody (HRP-conjugated anti-rabbit) for 1 h at room temperature. The experiment was repeated in triplicate. The bands were detected by enhanced chemiluminescence detection reagents (Applygen Technologies, Beijing, China).