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Figure 3 | BMC Microbiology

Figure 3

From: Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis

Figure 3

Cellular localization of FdhB (A), hydrogenase activity staining in native PAGE (B) and immunoblot with anti-FdhB (C). A. Immunoblotting of T. litoralis soluble fraction (S1), supernatant of the first (S2) and second (S3) wash of membrane fraction as well as the washed membrane fraction (WM). Proteins were separated on 12% SDS polyacrylamide gel after boiling in SDS-loading buffer for 10 min and the FdhB subunit was monitored using anti-FdhB antibody. One thousandth of the volume of the prepared fractions was loaded onto the gel to make the signals comparable. B. Proteins were separated on 6% native gel and stained for hydrogenase activity. S: soluble fraction M: unwashed membrane fraction WM: washed membrane fraction. The buffer used for the washing step contained 1% dodecyl-β-D-maltoside and 750 mM 6-aminohexanoic acid in 50 mM Bis-Tris pH 7.0. The unsoluble materials were removed by centrifugation at 50000 × g for 20 min and the supernatants were loaded onto the gel. C. The proteins from the activity stained gel were transferred to nitrocellulose membrane and screened with anti-FdhB antibody. The hydrogenase activity band corresponding to the FdhB signal is indicated with an arrow.

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