Study of lipid rafts by gel filtration combined with preliminary staining with fluorescently labeled antibodies

A novel procedure for detecting raft-associated proteins by gel filtration was developed. Cells were stained with fluorescently labeled protein-specific antibodies, lyzed in a nonionic detergent and gel-filtered on Sepharose 4B. Proteins were identified with the help of fluorescently labeled antibodies. Staining of cells with fluorescently labeled antibodies prior to cell lysis significantly simplified the identification procedure. The potentialities of the new approach are demonstrated in the example of several surface proteins associated with lipid rafts either constitutively or in the course of cell activation.[PUBLICATION ABSTRACT]