Jadwin Joshua A, Mayer Bruce J, Machida Kazuya
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.
Methods Mol Biol. 2015;1312:379-98. doi: 10.1007/978-1-4939-2694-7_38.
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.
远缘杂交印迹法是一种鉴定蛋白质-蛋白质相互作用的便捷方法，该方法将感兴趣的蛋白质样品固定在膜上，然后用非抗体蛋白进行检测。与使用特异性抗体检测目标蛋白的蛋白质印迹法不同，远缘杂交印迹法是根据蛋白质探针结合位点的有无来检测蛋白质。当使用特定的模块化蛋白质结合结构域作为探针时，这种方法可以鉴定参与信号转导等生物学过程的蛋白质-蛋白质相互作用，包括由翻译后修饰调节的相互作用。我们在此描述一种快速简便的远缘杂交印迹法方案，其中使用谷胱甘肽S-转移酶（GST）标签的Src同源2（SH2）结构域以磷酸化依赖的方式检测细胞蛋白。我们还提出了一种批量定量方法，可直接比较探针结合模式。
