Akkers Robert C, Jacobi Ulrike G, Veenstra Gert Jan C
Department of Molecular Biology, Nijmegen Center of Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
Methods Mol Biol. 2012;917:279-92. doi: 10.1007/978-1-61779-992-1_17.
Chromatin immunoprecipitation (ChIP) is a powerful technique to study epigenetic regulation and transcription factor binding events in the nucleus. It is based on immune-affinity capture of epitopes that have been cross-linked to genomic DNA in vivo. A readout of the extent to which the epitope is associated with particular genomic regions can be obtained by quantitative PCR (ChIP-qPCR), microarray hybridization (ChIP-chip), or deep sequencing (ChIP-seq). ChIP can be used for molecular and quantitative analyses of histone modifications, transcription factors, and elongating RNA polymerase II at specific loci. It can also be applied to assess the cellular state of transcriptional activation or repression as a predictor of the cells' capabilities and potential. Another possibility is to employ ChIP to characterize genomes, as histone modifications and binding events occur at specific and highly characteristic genomic elements and locations. This chapter provides a step-by-step protocol of ChIP using early Xenopus embryos and discusses potential pitfalls and other issues relevant for successful probing of protein-genome interactions by ChIP-qPCR and ChIP-seq.
染色质免疫沉淀（ChIP）是一种用于研究细胞核中表观遗传调控和转录因子结合事件的强大技术。它基于对体内已与基因组DNA交联的表位进行免疫亲和捕获。通过定量PCR（ChIP-qPCR）、微阵列杂交（ChIP-chip）或深度测序（ChIP-seq），可以获得表位与特定基因组区域关联程度的读数。ChIP可用于对特定基因座处的组蛋白修饰、转录因子和延伸中的RNA聚合酶II进行分子和定量分析。它还可用于评估转录激活或抑制的细胞状态，作为细胞能力和潜力的预测指标。另一种可能性是利用ChIP来表征基因组，因为组蛋白修饰和结合事件发生在特定且具有高度特征性的基因组元件和位置。本章提供了使用非洲爪蟾早期胚胎进行ChIP的详细步骤方案，并讨论了通过ChIP-qPCR和ChIP-seq成功探测蛋白质-基因组相互作用的潜在陷阱及其他相关问题。
