Association of EntB/G, -E, and -F with membranes. Total membranes were isolated from mid-log-phase E. coli AB1515 by the procedure of Osborn et al. (26). Membranes were resuspended in 65% sucrose, layered with decreasing concentrations of sucrose solutions (56, 53, 50, 47, 44, 41, 38, 35, and 28%), and separated as described by Poquet et al. (27). Membrane fractions were pooled, and 20 μg of protein (4) from each pool was analyzed by Coomassie blue staining (A) and Western blotting (B) after electrophoresis on 11% polyacrylamide gels (17). Lanes: 1, inner membrane fractions; 2, intermediate-density fractions; 3, light outer membrane fractions; 4, heavy outer membrane fractions; St, molecular weight standards.