Protein Coimmunoprecipitation in Arabidopsis
This protocol was adapted from “How to Study Gene Expression,” Chapter 7, in Arabidopsis by Detlef Weigel and Jane Glazebrook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.INTRODUCTION
Coimmunoprecipitation is one of the most useful techniques for revealing protein-protein interactions. Briefly, it can be divided into three steps: (1) preparation of cell extracts without disruption of protein interactions; (2) formation of the antibody-antigen complexes; and (3) affinity purification of the protein complexes and subsequent detection. Good negative controls to verify the specificity of the coimmunoprecipitation procedure are (1) performing the same immunoprecipitation experiment using beads coupled to the preimmune serum and (2) probing the Western blot with antibodies against protein known not to interact with the coimmunoprecipitated proteins under physiological conditions.
