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Journal of Lipid Research, Vol. 46, 1061-1067, May 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology
Methods |
A simplified method for the preparation of detergent-free lipid rafts
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110
1 To whom correspondence should be addressed. e-mail: pike{at}wustl.edu
Lipid rafts are small plasma membrane domains that contain high levels of cholesterol and sphingolipids. Traditional methods for the biochemical isolation of lipid rafts involve the extraction of cells with nonionic detergents followed by the separation of a low-density, detergent-resistant membrane fraction on density gradients. Because of concerns regarding the possible introduction of artifacts through the use of detergents, it is important to develop procedures for the isolation of lipid rafts that do not involve detergent extraction.
We report here a simplified method for the purification of detergent-free lipid rafts that requires only one short density gradient centrifugation, but yields a membrane fraction that is highly enriched in cholesterol and protein markers of lipid rafts, with no contamination from nonraft plasma membrane or intracellular membranes.
Abbreviations: CHO, Chinese hamster ovary; EGF, epidermal growth factor; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol
Supplementary key words caveolin • cholesterol • epidermal growth factor receptors • flotillin • Gq
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