Abstract
Peripheral proteins transiently interact with cellular membranes where they regulate important cellular events such as signal transduction. A number of peripheral proteins harbor lipid-binding modules that not only bind selectively with nanomolar affinity to biological membranes but also oligomerize on the membrane surface. In some cases, specific lipid binding or specific lipid compositions can induce peripheral protein oligomerization on cellular membranes. These oligomers serve different roles in biological signaling such as regulating protein-protein interactions, induction of membrane bending, or facilitating membrane scission. A number of technologies have been employed to study protein oligomerization with fluctuation analysis of fluorescently labeled molecules recently developed for use with commercial laser-scanning microscopes. In this chapter, the approach of raster image correlation spectroscopy coupled with number and brightness (N&B) analysis to investigate protein oligomerization on cellular membranes in live cells is presented. Important considerations are discussed for designing experiments, collecting data, and performing analysis. N&B analysis provides a robust method for assessing membrane binding and assembly properties of peripheral proteins and lipid-binding modules.