Western blot detect proteins expression levels

Left ventricular myocardial tissue samples were added with 500 µL of precooled lysate, ground, homogenized, lysed on ice and centrifuged to extract tissue protein. Use the BCA method to determine the protein level quantitatively. SDS-PAGE electrophoresis was conducted with 50 µg as the loading amount per well, electrotransferred to PVDF membrane, adding 5% skimmed milk powder solution, blocking at 37 °C for 1 hour, adding diluted recombinant anti-Bcl-2 recombinant anti-Bax, anti-Caspase-3 and anti-P53 antibody (1:2,000), recombinant anti-β-actin antibody (1:10,000), incubated overnight at 4 °C, washed 4 times with TBST, 10 min each time. Add diluted HRP-labeled goat anti-rabbit IgG antibody (1:2,000), incubate at 37 °C for 2 h, wash 4 times with TBST, 10 min each time. After treatment with a supersensitive luminescent liquid Super-GL ECL, it was exposed, developed, and fixed in a dark room. After taking pictures with the American Bio-Rad gel imaging analysis system, β-actin was used as an internal reference. Analyze with Gel-Pro Analyzer software and calculate the ratio of the gray value of Bcl-2, Bax, Caspase-3 and P53 to the gray value of β-actin as relative levels of protein expression.