Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.
Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.
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DOI：
10.1007/978-1-4939-6762-9_17
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发表时间：
2017
影响因子：
--
通讯作者：
K. Machida;Bernard A. Liu
中科院分区：
文献类型：
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作者：
K. Machida;Bernard A. Liu
SH2结构域对含磷酸酪氨酸序列的识别赋予酪氨酸激酶途径的特异性。通过评估分离的SH2结构域和它们的结合蛋白之间的相互作用，有可能深入了解否则难以接近的复杂细胞系统。远Western、下拉和荧光偏振（FP）已被频繁地用于表征磷酸酪氨酸信号传导。在这里，我们概述了这些已建立的测定使用重组SH2域的标准协议，强调适当的样品制备和测定控制的重要性。
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.