Membrane flotation on continuous sucrose gradients with Triton X-100.

Protein extracts were prepared as described above. The soluble and insoluble fractions were then separated by centrifugation at 15,000 × g for 15 min. The supernatant was discarded, and the insoluble-pellet fraction was resuspended in extraction buffer (as specified above) containing 1% Triton X-100 and then incubated for 20 min on ice. To remove gross insoluble material, the resulting extract was separated by centrifugation at 3,000 × g for 10 min. The pellet was discarded, and the supernatant was collected for further analysis. Samples (0.5 ml) were mixed with an equal volume of 90% sucrose in TE buffer. The 1-ml fractions of 45% sucrose-sample mixtures were overlaid with 1.2 ml of 35% sucrose, 1 ml of 30% sucrose, 1 ml of 25% sucrose, and 1 ml of 5% sucrose in TE. Membranes were separated by centrifugation using a Beckman SW-55 rotor for 16 h at 48,000 rpm (218,300 × g). Fractions (0.4 ml) were collected from the tops of the gradients and diluted in 1 ml of TE. The diluted fractions were precipitated by centrifugation at 100,000 × g for 30 min. Equal volumes of protein extracts from the different fractions were resolved by SDS-PAGE and analyzed by immunoblotting as described above.