Detection of His6-tagged Proteins by Western Blotting

Western blotting analysis was performed for detection of His₆-tagged proteins as previously described (Dong et al., 2012 (link)). Cells entering the stationary phase were harvested by centrifugation, washed with PBS (pH 7.0), resuspended in the same buffer, and sonicated. The cell lysates containing the same amount of proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, 10%). Proteins were then electrophoretically transferred to polyvinylidene difluoride membranes according to the manufacturer’s instructions (Bio-Rad). The gels were blotted for 2 h at 60 V using a Criterion blotter (Bio-Rad). The blotting membrane was probed with a rabbit polyclonal antibody against His₆-tag. Goat anti-rabbit IgG-HRP (horseradish peroxidase) (Roche Diagnostics) was used as the secondary antibody (1:5,000) and the signal was detected using a chemiluminescence Western blotting kit (Roche Diagnostics). Images were visualized with a UVP imaging system.