Anti rabbit igg conjugated to horseradish peroxidase

Western blotting was performed according to the guidelines in a previous report (Jensen, 2012) with some modifications. Briefly, cellular protein was extracted from mouse liver using lysis buffer, and protein concentration was determined with a BCA protein assay kit (KeyGEN, China). Denatured protein was separated on a 12% SDS‐PAGE along with 170 kDa to 10 kDa as molecular weight markers (Prestained Dual Color Protein Molecular Weight Marker, Beyotime, China). To save antibodies and improve the efficiency of immunoblotting, the part of the gel containing protein of the molecular weights of interest (10–17 kDa) was selected and cut, and then transferred to a polyvinylidene difluoride membrane (PVDF) (Beyotime, China). The PVDF membrane was blocked overnight in 5% skim milk in Tris‐buffered saline and Tween 20 (TBST) (10 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.1% Tween 20). For immunoblotting, the PVDF membrane was incubated for 2 hr with the antibody against LC3A (AP1805a, 1:500 dilution). Then, it was washed by TBST and incubated with anti‐rabbit‐IgG conjugated to horseradish peroxidase (1:5,000 Dako) for 1 hr. Finally, bands were visualized with X film (Fuji, Japan) by ECL‐Plus detection reagents (Santa Cruz, USA).