Staining comparison using hydrogen or formate as electron donor and different redox dye acceptors identifies Hyd-3 activity. Extracts from the strains MC4100, DHP-F2 (ΔhypF), FTD22 (ΔhyaB), FTD67 (ΔhybC), CP971 (ΔhycA-I), CP734 (ΔhyaB hybC), FTD147 (ΔhyaB hybB hycE), FTD150 (ΔhyaB hybC hycE hyfB-R), FM460 (ΔselC), FM911 (ΔfdhF), CPD17 (ΔhyaB hybC fdhE), CPD23 (ΔhyaB hybC fdhE fdhF) and CPD24 (ΔhyaB hybC fdoG fdnG) that were grown anaerobically in TGYEP media, pH 6.5 were used and 25 μg of protein were applied to non-denaturating PAGE (7.5% w/v polyacrylamide) and stained as indicated with either A: BV and TTC under a 100% hydrogen atmosphere, B: PMS and NBT under a 100% hydrogen atmosphere, or with C: BV, TTC and formate under 100% nitrogen atmosphere. In the interest of clarity only the genotypes of the strains are given. On the right hand side of the figure the migration patterns of hydrogenase 1 (Hyd-1), Hyd-2 and the mixed species of Fdh-N and Fdh-O (Fdh-N/O) are indicated, as well as the presumed migration of active FHL (Hyd-3). The top of each gel is marked by an arrow.