Preparation and analysis of protein extracts to investigate CCM pathogenesis

Abstract

Molecular techniques allow for the rapid discovery of CCM-associated protein targets, crucial to understanding CCM pathogenesis. Here, we describe optimized protein extraction methods that allow for extraction from whole cell, and/or cellular sub-compartments, including nuclear, mitochondria, cytoplasmic, and membrane-bound proteins, from lysates. This allows for the analysis of in vitro co-immunoprecipitation (Co-IP), label-free measurement of protein–protein interactions, multiplex protein–lipid binding assays, and western blots. Together, all these methods allow for a global analysis of the molecular mechanisms underlying CCM pathogenesis.