Diaminobenzidine reagent

Samples (40 μl of protein and 10 μl loading buffer) were boiled for 10 min. Protein samples were separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane for 35 min in an electrophoretic transfer cell (Bio-Rad Laboratories). Membranes were washed three times for 5 min in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% [v/v] Tween 20, pH 7.4), blocked with 5% skim milk for 2 h, and then incubated overnight at room temperature with rabbit serum (diluted 1:100 with 0.01 M PBS). Next, the membranes were washed four times for 5 min each in TBST, then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (diluted 1:1000) for 2 h. The membranes were washed four times with PBS, and protein signals detected using diaminobenzidine reagent (TIANGEN, Beijing, China).

Samples (40 μL of protein and 10 μL loading buffer) were boiled for 10 min, separated by 12% SDS-PAGE, and transferred to a nitrocellulose membrane over 35 min using a trans-blot SD semi-dry transfer cell (Bio-Rad, USA) at room temperature. Membranes were washed three times for 5 min each time in Tris-buffered saline-Tween- (TBST) (20 mM TRIS-HCl, 150 mM NaCl, 0.05% v/v tween-20, pH 7.4), coated in 5% skimmed milk for 2 h, then incubated overnight with positive serum (diluted 1:100 with 0.01 M PBS). Next, membranes were washed four times for 5 min each time in TBST, then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Earthox, USA) (diluted 1:1000) for 2 h. Membranes were washed four more times, and signals were detected using diaminobenzidine reagent (TIANGEN, Beijing, China).