Review for Anti-GAPDH antibody - Loading Control

Hepatocytes were washed with cold phosphate-buffered saline (PBS), collected in lysis buffer (Cell Signaling Technology), sonicated, and centrifuged at 16,000g for 15 mins, after which the supernatant was collected. For in vivo experiments, snap-frozen liver (median lobe) was homogenized in lysis buffer and centrifuged at 16,000g for 15 mins, after which the supernatant was collected. Protein concentrations were determined with the BCA (bicinchoninic acid) protein assay kit (Thermo Fisher Scientific). Loading buffer was added to the samples, which were then resolved by 10 or 15% SDS–polyacrylamide gel electrophoresis. Samples were then transferred onto a polyvinylidene difluoride membrane at 250 mA for 2 hours. The membrane was blocked in 5% milk for 1 hour and then incubated overnight with primary antibody in 1% milk. Membranes were washed in tris-buffered saline containing Tween (TBS-T) for 10 min, incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour, and then washed for 1 hour in TBS-T, before being developed for chemiluminescence (Thermo Fisher Scientific). The primary antibody anti-GAPDH was diluted at 1:1000, Abcam.