Isolation of lipid-rafts compartment by sucrose-gradient centrifugation

Detergent-resistant membrane (DRM) was isolated based on the standard protocol described previously [52–55]. Mouse cortical tissues or MDA-MB-231 cell line were lysed with cold tissue homogenization buffer [150 mM NaCl, 20 mM Na₂HPO₄, 2 mM NaH₂PO₄, 20% (v/v) glycerol, 2 mM sodium orthovanadate and protease inhibitors (Roche, Cat# 04 693 159 001, lot# 37536800), pH 7.4)] by 30 strokes in a Dounce homogenizer, followed by 20 passages through a 22-gauge needle. Then centrifuged for 11 min at 12,000×g in 4 °C to clear cellular debris and nuclear material. The supernatant was centrifuged at 124,000×g (SW55 rotor) for 90 min at 4 °C to pellet the total PM. The pellet was resuspended in 2 mL cold solubilizing buffer containing 0.5% v/v Triton X-100 in Mes-Buffered Saline (MBS, 25 mM MES, pH 6.5, 150 mM NaCl), protease inhibitors and 2 mM sodium orthovanadate and incubated for 30 min at 4 °C. Incubation time is important for this step to decrease contamination from other subcellular organelles, including, endoplasmic reticulum and mitochondria [56]. Two mL of solubilized PM was mixed with 2 mL of 80% (w/v) sucrose and applied to the bottom of a 12 mL ultracentrifuge tube. The 30% sucrose was layered on top, followed by 4 mL of MBS buffer containing 5% sucrose. The sucrose gradient was centrifuged at 164,000 xg (SW41Ti rotor) for 16 h at 4 °C to isolate the lipid raft and non-raft compartments. Twelve equal fractions (1 mL each) were collected from the top of gradient to the bottom. A creamy white layer at the 5–30% interface was identified and collected as lipid raft fraction.