FIG. 4.
Membrane association of the ArcB-MalF hybrid proteins. Cultures grown aerobically in Luria-Bertani broth were harvested during mid-exponential growth. The cells were washed with buffer S (50 mM Na-phosphate [pH 7.8], 300 mM NaCl, and 1 mM EDTA) by centrifugation. The cell pellet was resuspended in 3 ml of the same buffer and disrupted by sonication. Cell debris was removed by centrifugation for 10 min at 4,000 × g. The supernatant fluid was again centrifuged for 45 min at 35,000 × g to separate the cytosolic (C) and the membrane (M) fractions. The resultant supernatant fluid containing the soluble proteins was collected. The remaining pellet was resuspended in 0.5 ml of buffer M (20 mM HEPES [pH 7.5], 50 mM KCl, 1 mM EDTA, and 50% glycerol). Samples of cytosolic and membrane (containing 10 μg of protein) fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide gel) and the proteins were transferred to a Hybond-ECL filter (Amersham). The filter was equilibrated in TTBS buffer (25 mM Tris, 150 mM NaCl, and 0.05% Tween-20) for 10 min and incubated in blocking buffer (0.5% bovine serum albumin in TTBS) for 1 h at 37°C. ArcB polyclonal antibodies raised against His6-ArcB78-520 were added at a dilution of 1:10,000 to the filter and incubated for 1 h at room temperature. The bound antibody was detected by using anti-rabbit immunoglobulin G antibody conjugated to horseradish peroxidase and the ECL detection system (Amersham). The topology of the chimeric proteins is depicted: solid segments represent ArcB sequences, and hatched segments represent MalF sequences.