基于邻近连接的溶液内SH2结构域结合分析

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

作者信息

Machida Kazuya

机构信息

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.

出版信息

Methods Mol Biol. 2017;1555:331-347. doi: 10.1007/978-1-4939-6762-9_18.

DOI:10.1007/978-1-4939-6762-9_18

PMID:28092041

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.

摘要

由SH2结构域介导的蛋白质-蛋白质相互作用在酪氨酸激酶信号通路中赋予特异性。用于评估SH2结构域与其相互作用蛋白之间相互作用的传统检测方法，如远缘Western印迹法和下拉实验，本质上通量较低。我们开发了SH2-PLA，一种溶液内SH2结构域结合检测方法，它利用了邻近连接和实时PCR的速度和灵敏度。SH2-PLA仅使用几微升细胞裂解液就能快速评估SH2结构域与靶蛋白的结合，从而使其成为研究酪氨酸激酶信号传导的一种有吸引力的新工具。