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Lipid rafts and their related membrane vesicular structures, caveolae, are cholesterol- and sphingolipid-rich microdomains of the plasma membrane that have attracted considerable interest because of their ability to concentrate numerous signaling proteins. Efforts to define the proteins that reside in lipid rafts and caveolae as well as investigations into the functional role of these microdomains in signaling, endocytosis, and other cellular processes have led to the hypothesis that they compartmentalize or prearrange molecules involved in regulating these pathways. This chapter describes biochemical approaches for defining lipid rafts and caveolae. Included are detergent- and nondetergent-based fractionations on sucrose-density gradients that isolate buoyant lipid rafts and caveolae as well as caveolin antibody-based immunoisolation of detergent-insoluble membranes that selectively isolates caveolae and not lipid rafts. Also, a general method to disrupt lipid rafts and caveolae using β-cyclodextrin that is useful for probing the role of these microdomains in cellular processes is described. The advantages and disadvantages of the respective approaches are discussed. Taken together, these methods are useful for defining the role of lipid rafts and caveolae in cell signaling.
