Anti histidine hrp conjugated antibody

The purified proteins were analyzed through 8% SDS-PAGE and western blotting using a prestained high molecular weight protein ladder (Fermentas, Lithuania). The protein concentration was measured using Bradford method with bovine serum albumin (BSA) as the standard protein. For western blotting, proteins were transferred on the nitrocellulose membrane in a wet blotting system (Denagen Tajhiz, Iran). The membrane was blocked with 2% (w/v) skimmed milk (Sigma, USA) in PBS buffer (137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1 mM KH₂PO₄, pH7.4) at 4 °C for 16 h (overnight). The membrane was washed four times with PBS buffer containing 0.05% Tween-20 (PBST) and incubated with anti-Histidine HRP conjugated antibody (1:2000 dilution) (Sigma, USA) at room temperature for 1 h. The membrane was washed five times with PBST buffer and one more time with PBS buffer alone. At the end, the membrane was stained with 3,3′-diaminobenzidine (DAB) (Sigma, USA).