Analysis of Lipids, Fatty Acid, and Cholesterol in Membrane Microdomains.

The original concept that lipid and protein components are randomly distributed in cellular membranes has been challenged by evidence of compartmentalization of such components into discrete membrane microdomains (known as lipid rafts). The lipid microdomain hypothesis has generated significant controversy and rigorous inquiry to test the idea that such domains concentrate machinery to mediate cellular processes such as signaling, synaptic plasticity, and endocytosis. As such, a large number of studies have used biochemical, cell biological, and biophysical methodologies to define the composition of membrane microdomains in experimental contexts. Although biochemical preparation strategies are not without limitations (as discussed herein), the isolation of detergent-resistant and detergent-free membrane domains can provide important information about the segregation of lipids and proteins in membranes. In this chapter, we describe methodologies to isolate membranes from cell or tissue sources with biophysical/biochemical properties of membrane microdomains and also provide methods for subsequent classical or mass spectrometry-based lipid analytical approaches.