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In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

Methods Mol Biol.. 2017;

Machida K. 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.

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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.

Keywords

EGFR; Grb2; In-solution binding assay; Proximity ligation; Real-time PCR; SH2 domain

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