Characterizing Bcl-2 family protein conformation and oligomerization using cross-linking and antibody gel-shift in conjunction with native PAGE

Abstract

The Bcl-2 family of proteins tightly controls the intrinsic or mitochondrial pathway of apoptosis. This family is subdivided based on function into pro-survival proteins (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bfl -1/A1) and pro-apoptotic proteins. The pro-apoptotic subset is further divided into those proteins that initiate the pathway, the BH3-only proteins (including Bim, Puma, Noxa, and Bid), and those that execute the pathway, Bak and Bax. Whether a cell lives or dies in response to apoptotic stress is determined by the interactions of the Bcl-2 family, which is in turn influenced by their conformation. We describe here a protocol to interrogate the interactions and conformation of the Bcl-2 family of proteins under native conditions.