Figure 1

The detergent-free isolation protocol of lipid rafts. Six × 10⁷ YH16.33 cells were centrifuged at 250 g for 10 minutes and the pellet was re-suspended in a base buffer consisting of 20 mM Tris-HCl, 250 mM Sucrose(pH 7.8), supplemented with 1 mM CaCl₂ and 1 mM MgCl₂ . The cells were then lysed by passing through a 23 gauge needle 20 times. The lysates were centrifuged at 1000 rpm for 10 minutes and the supernatant was saved. The pellet was re-suspended in fresh base buffer and then again passed though a 23 gauge needle 20 times. After centrifugation at 1000 rpm for 10 minutes again at 4°C, the supernatant was collected and pooled with the previously collected supernatant. An OptiPrep (Sigma-Aldrich, St. Louis, MO) gradient was prepared with a final 25% OptiPrep dilution at the bottom of an ultracentrifuge tube and overlaid with 20%, 15%, 10%, 5% and 0% OptiPrep solutions. The gradient was centrifuged at 52,000 × g for 90 minutes at 4°C and nine 1.3 ml aliquots from the top of gradient were collected and stored at -20°C until further analysis.