Wiehle Laura, Breiling Achim
Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
Methods Mol Biol. 2016;1480:7-21. doi: 10.1007/978-1-4939-6380-5_2.
Chromatin immunoprecipitation (ChIP) is a valuable method to investigate protein-DNA interactions in vivo. Since its discovery it has been indispensable to identify binding sites and patterns of a variety of DNA-interacting proteins, such as transcription factors and regulators, modified histones, and epigenetic modifiers. The Polycomb repressors were the first proteins that have been mapped using this technique, which provided the mechanistic basis for the understanding of their biological function. Cross-linked (XChIP) or native (NChIP) chromatin from tissues or cultured cells is fragmented and the protein of interest is immunoprecipitated using a specific antibody. The co-precipitated DNA is then purified and subjected to analysis by region-specific PCR, DNA microarray (ChIP-on-chip), or next-generation sequencing (ChIP-seq). The assay can therefore produce information about the localization of the analyzed protein at specific candidate loci or throughout the entire genome. In this chapter, we provide a detailed protocol of the basic standard ChIP assay and some remarks about variations.
染色质免疫沉淀法（ChIP）是一种在体内研究蛋白质与DNA相互作用的重要方法。自发现以来，它对于鉴定各种与DNA相互作用的蛋白质（如转录因子和调节因子、修饰的组蛋白以及表观遗传修饰因子）的结合位点和模式而言不可或缺。多梳抑制因子是首批使用该技术进行图谱绘制的蛋白质，这为理解其生物学功能提供了机制基础。来自组织或培养细胞的交联（XChIP）或天然（NChIP）染色质被片段化，然后使用特异性抗体免疫沉淀感兴趣的蛋白质。随后纯化共沉淀的DNA，并通过区域特异性PCR、DNA微阵列（芯片上的ChIP）或新一代测序（ChIP-seq）进行分析。因此，该检测可以产生有关所分析蛋白质在特定候选基因座或整个基因组中的定位信息。在本章中，我们提供了基本标准ChIP检测的详细方案以及关于变体的一些说明。
