Separation of inner and outer membranes by sucrose floatation gradient.

Cells were collected by centrifugation and resuspended in 25 mM HEPES, pH 7.4, plus Complete EDTA-free protease inhibitor cocktail (Roche). Cells were disrupted by two passages through an LV1 Microfluidizer homogenizer (Microfluidics). The cell lysate was supplemented with 10 μg/ml of DNase I, and unbroken cells were removed by centrifugation at 4,000 × g. Membranes were collected by ultracentrifugation at 260,000 × g for 1 h and washed once with HEPES buffer. Membranes were resuspended in 25 mM HEPES, pH 7.4, and sucrose was dissolved to achieve >60% (wt/wt) saturation. The inner and outer membranes were separated by floatation in a sucrose gradient for 40 h at 230,000 × g at 10°C as described elsewhere (43). Fractions were removed and analyzed by SDS-PAGE and immunoblotting with appropriate antibodies.