His-Tag Western blotting

Protein and solubilised outer and inner membranes were subject to a 12% (w/v) SDS-PAGE, followed by electrophoretic transfer to a PVDF membrane with transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) by using Trans-Plot SD Cell (Bio-Rad, Hercules, CA). His-Tag Western blot was carried out using the Pierce Fast Western Blot Kit (Thermo Scientific, Waltham, MA). Blotted membrane has been placed in the wash blot solution Fast Western 1× Wash Buffer to remove transfer buffer. Primary Antibody Working Dilution was added to the blot and incubated for 30 min at room temperature (RT) with shaking. After, the blot was removed from the primary antibody solution and incubated for 10 min with the Fast Western Optimised HRP Reagent Working Dilution. Subsequently, the membrane was washed two times in about 20 ml of Fast Western 1× Wash Buffer. Finally, the membrane was incubated with the detection reagent working solution and incubated for 3 min at room temperature and then developed with X-ray film.