Anti 6 his tag mouse monoclonal antibody

Western blotting was applied to conform the His-tag fused to rIL-17RC proteins. Initially, on completion of separation using 10% SDS-PAGE, the purified proteins were blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore), and subsequently 5% nonfat dry milk for blocking step. After that, overnight incubation in anti-6 × His Tag mouse monoclonal antibody (Sangon, Shanghai, China) at a dilution of 1:5000 at 4°C was performed. Following repeated washes with TBS-Tween buffer for 4 times, HRP-conjugated goat antimouse IgG (1:8000; Sangon, Shanghai, China) were used as secondary antibody. After thorough washing, the membrane was detected with a highly sensitive ECL luminescence reagent (Sangon, Shanghai, China) according manufacturer’s instructions.

The purified protein was separated using 10% SDS–PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (100 V, 1 h). The membrane was treated with 5% blocking protein powder in TBST (1 M Tris-HCl pH 7.5, 500 mM NaCl, and 0.2% Tween 20) for 1 h at room temperature and reacted with the anti-6 × His tag mouse monoclonal antibody (Sangon, Shanghai, China) in TBST for 1.5 h at room temperature. The membrane was washed with TBST 4 times for 5 min each time and then incubated with AP-conjugated goat anti-mouse IgG (Sangon, Shanghai, China) in TBST for 1 h at room temperature. The NBT/BCIP substrate solution (Sangon, Shanghai, China) was utilized to visualize the protein band after the PVDF membrane was washed again.