Analysis of Protein-Protein Interaction by Co-IP in Human Cells
Analysis of Protein-Protein Interaction by Co-IP in Human Cells
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DOI：
10.1007/978-1-4939-7871-7_20
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发表时间：
2018-01-01
期刊：
影响因子：
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通讯作者：
Takahashi, Yoshinori
中科院分区：
文献类型：
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作者：
Tang, Zhenyuan;Takahashi, Yoshinori
虽然有各种方法可用于分析蛋白质-蛋白质相互作用，但免疫共沉淀（co-IP）仍然是发现新的蛋白质相互作用或确定蛋白质物理缔合的最经典和最常用的方法之一。该测定开始于在适当的裂解缓冲液中制备总细胞或组织裂解物。使用特异性抗体捕获裂解物中的目标蛋白质，并使用树脂使其与其结合蛋白一起沿着沉淀。在一系列洗涤以去除裂解物中未结合的蛋白质后，对所得免疫复合物进行免疫印迹、凝胶内蛋白质染色或质谱分析以确定感兴趣的蛋白质-蛋白质相互作用。在本章中，描述了一个标准的IP/co-IP协议，并讨论了潜在的问题和故障排除。
While there are various approaches available to analyze protein-protein interactions, coimmunoprecipitation (co-IP) remains one of the most classic and commonly used methods to discover novel protein interactions or to determine the physical association of proteins. The assay begins with the preparation of total cell or tissue lysate in an appropriate lysis buffer. Protein of interest in the lysate is captured using a specific antibody and precipitated along with its binding proteins using a resin. After a series of washes to remove nonbound proteins in the lysate, the resultant immune complexes are subjected to immunoblotting, in-gel protein staining, or mass spectrometry to determine the protein-protein interaction of interest. In this chapter, a standard IP/co-IP protocol is described and potential problems and troubleshooting are discussed.