Horseradish peroxidase labeled goat anti rabbit igg

Cells were harvested after treatment and lysed with RIPA lysis buffer (P0100; Solarbio, Beijing, China) containing 1 mm phenylmethane on ice for 5 min. After centrifugation at 12000 rpm and 4°C for 10 min, the supernatant was collected to obtain total protein. The concentration of proteins was determined using a BCA protein assay kit (PC0020; Solarbio, Beijing, China). According to the Western blot protocol, proteins were loaded and separated on 8% SDS-PAGE and then transferred onto a PVDF membrane (IPVH00010; Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat milk, and the anti-Bcl-2 primary antibody (diluted 1:500; WL01556; Wanleibio, Shenyang, China) was added and incubated at 4°C overnight. After washing with TBST, the membrane was incubated with horseradish peroxidase-labeled goat anti-rabbit igg (diluted 1:3000; SE134; Solarbio). The internal reference β-actin was similarly processed. Finally, ECL solution (PE0010; Solarbio) was added to the membrane followed by exposure to X-ray film in a dark room. The film was developed and scanned, and the optical density value of the target band was analyzed with a gel image processing system (Gel-Pro-Analyzer software).