使用重组SH2结构域的结合分析：Far-Western、下拉和荧光偏振分析。

Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

作者信息

Machida Kazuya, Liu Bernard

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, 06030, USA.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

出版信息

Methods Mol Biol. 2017;1555:307-330. doi: 10.1007/978-1-4939-6762-9_17.

DOI:10.1007/978-1-4939-6762-9_17

PMID:28092040

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.

摘要

SH2结构域对含磷酸酪氨酸序列的识别赋予了酪氨酸激酶信号通路的特异性。通过评估分离的SH2结构域与其结合蛋白之间的相互作用，有可能深入了解原本难以触及的复杂细胞系统。Far-Western、下拉法和荧光偏振（FP）已被频繁用于磷酸酪氨酸信号转导的表征。在这里，我们概述了使用重组SH2结构域进行这些既定检测的标准方案，强调了适当样品制备和检测对照的重要性。