Lipid Raft Isolation by Sucrose Density Gradient Centrifugation

Low-density membrane raft fractions were isolated as described previously (Bourova et al., 2003) using sucrose density gradient centrifugation. MOR-CHO cells were washed in ice-cold phosphate-buffered saline (pH 7.3), harvested and homogenized in 50 mM Tris buffer (pH 7.4) containing 3 mM MgCl2, 1 mM EDTA, 1 mM PMSF and complete cocktail protease inhibitor tablet (1/50ml). Exactly 2 mL of the resulting homogenate was mixed with 2 mL of ice-cold 80% w/v sucrose in 50 mM Tris buffer (pH 7.4), transferred into a centrifuge tube for a Beckman ultracentrifuge SW 41 rotor and overlaid very slowly with 35%, 30%, 25%, 20% 15%, 10% (1 mL each) and 5% w/v (1.5 mL) sucrose in 50 mM Tris buffer (pH 7.4). Great care was devoted to prevent mixing of the layers. Following centrifugation at 188,000 g for 20 h, density gradient fractions (first 0.5 ml followed by eleven aliquots of 1 ml fractions) were collected from the meniscus using an Auto Densi-Flow density gradient fractionator (Labconco, Kansas city, MO). Aliquots were snap frozen and stored at −80°C until further use.