Western Blot Analysis of Polyhistidine-Tagged Proteins

The purified protein was run in SDS-PAGE (12 %), followed by blotting onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 3 % BSA in 1X TBST buffer (pH 7.6). The membrane was washed three times with 1X TBST buffer. Then the membrane was incubated with monoclonal anti-polyhistidine antibody (Sigma, St. Louis, MO, USA) at 4 °C for 12 h, followed by thorough washing with 1X TBST buffer. After this, anti-mouse IgG alkaline phosphatase (secondary antibody) (Sigma, St. Louis, MO, USA) was introduced, and incubated at room temperature for 2 h. After washing with 1X TBST buffer, the membrane protein was visualized by 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium) substrate (Sigma, St. Louis, MO, USA) at room temperature [37 , 69 (link)].