Western blot analysis of expression of SH hydrogenase. FTD147/pJWPH5) was cultured overnight at 30°C in LB medium with 0.05 mM IPTG under anaerobic conditions. The culture was harvested by centrifugation, sonicated, and centrifuged (15 min, 10,000 rpm). 25–35 μg pellet (lane 2) and supernatant (lane 3) were electrophoresed on 12% SDS-polyacrylamide gels (Laemmli and Favre (1973)), transferred to PVDF membrane, developed with primary anti-serum to SH hydrogenase, and revealed by chemiluminescence as previously described (Yakunin and Hallenbeck (1998)). A similarly cultured and prepared extract of FTD147 was also analyzed (supernatant, lane 4, pellet, lane 5) and found to be devoid of these protein bands. As a positive control, an aliquot of the supernatant of a 45 min 100,00 g centrifugation of a sonicated extract of R. eutropha H16 grown anaerobically overnight on NB medium at 30°C was also loaded (lane 1).