Detection of Protein-Protein Interactions by Far-Western Blotting
Detection of Protein-Protein Interactions by Far-Western Blotting
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DOI：
10.1007/978-1-59745-542-8_34
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发表时间：
2009-01-01
期刊：
影响因子：
--
通讯作者：
Mayer, Bruce J.
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文献类型：
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作者：
Machida, Kazuya;Mayer, Bruce J.
远蛋白质印迹是一种表征蛋白质-蛋白质相互作用的便捷方法，在这种方法中，感兴趣的蛋白质样品不被固定在膜上，然后用非抗体蛋白质进行探测。与使用特定抗体来检测目标蛋白质的Western blotting不同，远Western blotting根据蛋白质探针结合位点的存在或不存在来检测蛋白质。当使用特定的模块化蛋白质结合域作为探针时，该方法可以表征生物过程中涉及的蛋白质-蛋白质相互作用，包括受翻译后修饰调控的相互作用。我们在这里描述了一种快速而简单的远蛋白质印迹方法，其中GST标记的Src同源2(SH2)结构域被用来以磷酸化依赖的方式探测细胞蛋白质。
Far-western blotting is a convenient method to characterize protein-protein interactions, in which protein samples of interest are unmobilized on a membrane and then probed with a nonantibody 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 the 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.