FIGURE 1.

Gel analysis of His-tagged complex I. A, immunoblotting analysis of E. coli membranes from wild type (lane 1) and the (His)₉-nuoE mutant (lane 2). Membranes (20 μg/lane) were loaded onto a 12.5% Laemmli SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred onto nitrocellulose membranes, and Western blotting was carried out using the SuperSignal West Pico system. Antibodies specific to NuoE and His tag were used. B, SDS-PAGE of purified complex I. Tricine SDS-PAGE on a 10% gel was performed according to Ref 40. C, Blue-Native PAGE of purified complex I (CI). The arrows show the location of the fully assembled complex I and NuoEFG subcomplex. Blue-Native gels were stained with Coomassie Brilliant Blue (left) or incubated with 2.5 mg/ml nitro blue tetrazolium and 150 μm NADH for 1 h at 37 °C (right).