Immunoblot polyvinylidene uoride pvdf membranes

Cardiomyocytes were washed twice with cold PBS, harvested, lysed in ice-cold lysis buffer (pH = 7.4, 100 mM KCl, 10 mM HEPES, 1.5 mM MgCl), sonicated and homogenized in RIRP buffer. After centrifugation, a Pierce BCA protein assay kit was used to determine the protein concentration. Equal concentrations of soluble lysate protein (50 mg) were added to a 12% SDS-PAGE Bis-Tris gel, and the protein was transferred to immunoblot polyvinylidene uoride (PVDF) membranes (Bio-Rad, USA). Membranes were incubated and blocked overnight at 4°C with antibodies against p-AMPK/AMPK, p-Akt/Akt and GAPDH.
Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution). The immunoreactive bands were analyzed with an enhanced chemiluminescence substrate kit (Biological Industries, BI, Israel).

Cardiomyocytes were washed twice with cold PBS, harvested, lysed in ice-cold lysis buffer (pH = 7.4, 100 mM KCl, 10 mM HEPES, 1.5 mM MgCl), sonicated and homogenized in RIRP buffer. After centrifugation, a Pierce BCA protein assay kit was used to determine the protein concentration. Equal concentrations of soluble lysate protein (50 mg) were added to a 12% SDS-PAGE Bis-Tris gel, and the protein was transferred to immunoblot polyvinylidene uoride (PVDF) membranes (Bio-Rad, USA). Membranes were incubated and blocked overnight at 4°C with antibodies against p-AMPK/AMPK, p-Akt/Akt and GAPDH. Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution). The immunoreactive bands were analyzed with an enhanced chemiluminescence substrate kit (Biological Industries, BI, Israel).