title: Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.
abstract: Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.
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. machida@uchc.edu.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA, USA.
authors.forename: Bernard
authors.initials: B
authors.lastname: Liu
mesh_terms: D015153:Blotting, Western / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods; D005455:Fluorescent Antibody Technique; D008163:Luminescent Measurements / Q000379:methods; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine
keywords: Far-Western; Fluorescence polarization; Pervanadate treatment; Pull-down; SH2 binding assay; SH2 domain