Purification of caveolae in gradients prepared from OptiPrep™

Early methods for the purification of lipid-rich plasma membrane domains largely relied on their insolubility in Triton X-110 (or some other non-ionic detergent) relative to that of the bulk plasma membrane or that of all the other subcellular membranes. 

Sometimes detergent was added to the whole homogenate or more frequently a partially-purified plasma membrane fraction was first isolated before treating with detergent. Smart et al [1] however pointed out that, while use of a non-ionic detergent did permit the isolation of a lipid-rich membrane domain, that some characteristic caveolar proteins can be lost in the procedure. 

These workers therefore developed a method that avoids the use of Triton X-100. After isolation of a plasma membrane fraction from either human skin fibroblasts or MA104 cells, the caveolae are released by sonication in a standard cell homogenization medium. 

The first part of the isolation procedure is a flotation through a continuous iodixanol gradient (0-20%); this gradient is essentially a resolving gradient in which the caveolinrich vesicles are concentrated in the top third of the gradient, while the predominantly caveolin-poor vesicles band in denser regions. A second discontinuous gradient is essentially a concentration gradient to band the caveolin-rich vesicles sharply at an interface.