title: Isolation of Lipid Rafts Through Discontinuous Sucrose Gradient Centrifugation and Fas/CD95 Death Receptor Localization in Raft Fractions.
abstract: Lipid raft domains, enriched in sphingolipids and cholesterol, serve as sorting platforms and hubs for signal transduction proteins, and show resistance to detergent solubilization. Despite rafts have been involved in survival processes, these membrane domains have also been shown to play a major role in the modulation of death receptor signaling. Here, we describe a detailed protocol for isolating lipid rafts from whole cells by taking advantage of the lipid raft resistance to Triton X-100 solubilization at 4 °C, followed by sucrose gradient centrifugation, with subsequent analysis of Fas/CD95 death receptor localization in the raft fractions by immunoblotting. This method is also useful to localize additional proteins in membrane rafts.
authors.affiliation: Laboratory of Cell Death and Cancer Therapy, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), C/ Ramiro de Maeztu 9, 28040, Madrid, Spain. cgajate@cib.csic.es.
authors.forename: Consuelo
authors.initials: C
authors.lastname: Gajate
authors.affiliation: Laboratory of Cell Death and Cancer Therapy, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), C/ Ramiro de Maeztu 9, 28040, Madrid, Spain. fmollin@usal.es.
authors.forename: Faustino
authors.initials: F
authors.lastname: Mollinedo
mesh_terms: D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D053222:Fas Ligand Protein / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D013395:Sucrose; D057188:Workflow; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D053222:Fas Ligand Protein; D019014:fas Receptor; D013395:Sucrose
keywords: Cell disruption; Cholera toxin B subunit; Death receptor; Detergent-resistant membrane; Fas/CD95; Immunoblotting; Lipid rafts; Sucrose gradient centrifugation; Triton X-100