Detection of protein-protein interactions by far-western blotting.
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 nonantibody 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 the 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.
Machida
Kazuya
K
Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, 06030, USA. machida@neuron.uchc.edu
Mayer
Bruce J
BJ
D001665:Binding Sites; D026481:Blotting, Far-Western / Q000379:methods*; D025941:Protein Interaction Mapping; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D011506:Proteins; D011993:Recombinant Fusion Proteins