Jedamzik Britta, Eckmann Christian R
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
Cold Spring Harb Protoc. 2009 Oct;2009(10):pdb.prot5299. doi: 10.1101/pdb.prot5299.
Protein coimmunoprecipitation (co-IP) is a method used to analyze in vivo complex formation of various proteins. Although such an analysis supports the coexistence of proteins in a complex, a direct protein-protein interaction cannot be concluded unless further in vitro data are available. This protocol describes how to perform co-IPs from C. elegans whole-worm extracts using protein-specific antibodies. First, we describe how to culture a large number of worms while maintaining their overall appearance and wild-type fertility rates, which are important factors when analyzing the germline tissue. Next, we present a gentle and effective method to generate worm extracts with high protein concentrations that maintain protein complexes of high quality. Finally, we describe how to purify the protein of choice along with its associated complex members. The precipitated protein complex can be analyzed by either immunoblot analysis or mass spectrometry to identify the copurified protein components. When working with RNA-binding proteins, it is of interest to assess whether RNA molecules, rather than a direct interaction between the proteins, might mediate complex formation. For this purpose, an optional RNase digestion step to degrade the RNA in the extract is described.
蛋白质免疫共沉淀（co-IP）是一种用于分析各种蛋白质在体内形成复合物的方法。尽管这种分析支持蛋白质在复合物中共存，但除非有进一步的体外数据，否则不能得出蛋白质-蛋白质直接相互作用的结论。本方案描述了如何使用蛋白质特异性抗体从秀丽隐杆线虫全虫提取物中进行免疫共沉淀。首先，我们描述了如何在保持其整体外观和野生型生育率的同时培养大量线虫，这在分析生殖系组织时是重要因素。接下来，我们介绍一种温和有效的方法来生成高蛋白浓度的线虫提取物，同时保持高质量的蛋白质复合物。最后，我们描述了如何纯化目标蛋白质及其相关的复合物成员。沉淀的蛋白质复合物可以通过免疫印迹分析或质谱分析来鉴定共纯化的蛋白质成分。当处理RNA结合蛋白时，评估RNA分子而非蛋白质之间的直接相互作用是否可能介导复合物形成是很有意义的。为此，描述了一个用于降解提取物中RNA的可选RNase消化步骤。
