title: Detection and quantification of protein-protein interactions by far-western blotting.
abstract: Far-western blotting is a convenient method to characterize protein-protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a non-antibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein-protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner. We also present a batch quantification method that allows for the direct comparison of probe binding patterns.
authors.affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, Farmington, CT, 06030, USA.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Jadwin
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Mayer
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
mesh_terms: D026481:Blotting, Far-Western / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D008567:Membranes, Artificial; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D055767:Immobilized Proteins; D008567:Membranes, Artificial; D011506:Proteins; D014443:Tyrosine