Cell-Free Antibody Capture Method for Analysis of Detergent-Resistant Membrane Rafts

Cholesterol-rich microdomains present on the plasma membrane appear to play an important role in spatio-temporal regulation of cell signaling and cell adhesion processes. Compositional heterogeneity of these microdomains and their coalescence during cell-cell interactions may provide one mechanism for triggering and/or regulating signaling cascades from the plasma membrane to the cell interior. Biochemical analyses of distinct lipid microdomain subpopulations and single-rafts obtained from unstimulated and ligand-stimulated cells are critical for deciphering functional role of lipid rafts. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Moreover, this cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts, and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signaling on a single lipid raft-basis.