title: In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.
abstract: Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.
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, 06032, USA. machida@uchc.edu.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins; D015203:Reproducibility of Results; D012996:Solutions; D018909:src Homology Domains
chemical_list: D000906:Antibodies; D011993:Recombinant Fusion Proteins; D012996:Solutions
keywords: EGFR; Grb2; In-solution binding assay; Proximity ligation; Real-time PCR; SH2 domain