Detection and Quantification of Protein-Protein Interactions by Far-Western Blotting
Detection and Quantification of Protein-Protein Interactions by Far-Western Blotting
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DOI：
10.1007/978-1-4939-2694-7_38
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发表时间：
2015-01-01
期刊：
影响因子：
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通讯作者：
Machida, Kazuya
中科院分区：
文献类型：
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作者：
Jadwin, Joshua A.;Mayer, Bruce J.;Machida, Kazuya
远蛋白质印迹是表征蛋白质-蛋白质相互作用的方便方法，其中感兴趣的蛋白质样品固定在膜上，然后用非抗体蛋白质进行探测。与使用特异性抗体来检测靶蛋白的蛋白质印迹法相反，远蛋白质印迹法基于蛋白质探针的结合位点的存在或不存在来检测蛋白质。当特定的模块化蛋白质结合结构域被用作探针时，这种方法允许表征参与生物过程如信号转导的蛋白质-蛋白质相互作用，包括由翻译后修饰调节的相互作用。在这里，我们描述了一个快速和简单的协议远Western印迹，其中GST标记的Src同源2（SH 2）结构域被用来探测细胞蛋白的磷酸化依赖的方式。我们还提出了一个批量定量方法，允许探针结合模式的直接比较。
Far-western blotting is a convenient method to characterize protein-protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a non-antibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein-protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner. We also present a batch quantification method that allows for the direct comparison of probe binding patterns.