Isolation and Fractionation of Cellular Membranes

Cells were harvested using trypsin, homogenized in homogenization buffer (250 mM sucrose, 20 mM HEPES-KOH, pH 7.4, 1 mM EDTA, and protease inhibitor cocktail), and centrifuged at 800 ×g for 10 min to remove the nuclei. The supernatant was centrifuged at 20,000 ×g for 30 min, and the pellet (membrane fraction) was resuspended in 19% (v/v) OptiPrep solution (Axis-Shield Density Gradient Media-Alere Technologies AS) as described previously.³⁰ (link)^,38 (link) The sample was loaded on a discontinuous density gradient with 27%, 22.5%, 19%, 16%, 12%, and 8% OptiPrep (2 mL each) in a 12-mL centrifugation tube and subjected to ultracentrifugation at 13,000 ×g for 4 h at 4°C in a swinging bucket rotor (SW 40.1; Beckman Coulter). Subcellular fractions were collected from the top of the tube, washed, concentrated in homogenization buffer, and centrifuged at 20,000 ×g for 30 min. The protein contents of the individual fractions were determined by western blotting.