Heme-Staining and Immunoblotting Analysis

Unless otherwise noted, cells of the late exponential phase were harvested, washed with phosphate buffered saline (PBS), resuspended in the same buffer, and sonicated. Protein concentrations of the cell lysates was determined by the bicinchoninic acid assay (Pierce Chemical). For heme-staining, the cell lysates were separated on SDS-PAGE using 12% polyacrylamide gels and stained with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as described elsewhere43 (link). Immunoblotting analysis was performed essentially the same as previously described44 (link). Proteins separated on SDS-PAGE were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane 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 rabbit polyclonal antibodies against NrfA. The 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) in accordance with the manufacturer’s instructions. Images were visualized with a UVP imaging system.