Abstract
Organelles physically interact with each other via protein tethering complexes that bridge the opposing membranes. Organelle membrane contacts are highly dynamic, implying dynamism of the tethering complexes. Alterations in the binding of the tethering proteins can be assessed by immunoprecipitation. Antibody-conjugated beads allow for purification of the target protein with its binding partners, which can subsequently be examined by western blot analysis. We present immunoprecipitation methods and strategies to examine protein interaction domains, and for the identification of residues important for the regulation of the interaction, here focusing on phosphorylation. We use the peroxisomal membrane protein ACBD5 and its paralog ACBD4, which both bind ER membrane protein VAPB to mediate peroxisome-ER contacts, as example. However, this method can be applied to other peroxisomal and non-peroxisomal (membrane) proteins.