Co-immunoprecipitation from Brain.

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 brief, this technique consists of immunocapturing a protein of interest from a tissue lysate using a specific antibody. Subsequently, the immunocomplex containing the antibody, the antigen of interest and the antigen-associated proteins is precipitated by means of a Proteins A/G conjugated resin and analysed by immunoblot. Of note, since PPIs are extremely dynamic and/or labile, the Co-IP method has to be optimized for each specific PPI. Remarkably, it is mandatory paying special attention to the specificity of antibodies used in the immunoprecipitation and the lysis buffer employed. Finally, it is important to point out that, by means of this approach, it is difficult to differentiate physiological (real) interactors from non-specific binders, thus an additional tool is recommended to validate the results. 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.