Co-Immunoprecipitation from Brain

Abstract

Co-immunoprecipitation (Co-IP) is one of the most broadly used methodologies to identify, validate, and characterize protein–protein interactions (PPIs) in native conditions. In this technique, a protein of interest is immunocaptured from a tissue lysate using a specific antibody. The immunocomplex containing the antibody, the antigen of interest, and the antigen-associated proteins is then precipitated by means of a protein A/G conjugated resin and analyzed by immunoblot. Interestingly, as PPIs may be extremely dynamic and/or labile, special attention to the antibodies and the lysis buffer used should be paid; therefore, the Co-IP method needs to be optimized for each specific PPI. Finally, an important question with this approach is the difficulty to differentiate physiological (real) interactors from nonspecific binders. Here, we describe the methodology to Co-IP two plasma membrane receptors from different brain areas (i.e., hippocampus and raphe nucleus) using the same set of antibodies.