Analysis of epigenetic modifications of chromatin at specific gene loci by native chromatin immunoprecipitation of nucleosomes isolated using hydroxyapatite chromatography
Analysis of epigenetic modifications of chromatin at specific gene loci by native chromatin immunoprecipitation of nucleosomes isolated using hydroxyapatite chromatography
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DOI：
10.1038/nprot.2008.8
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Dilworth, F. Jeffrey
中科院分区：
文献类型：
--
作者：
Brand, Marjorie;Rampalli, Shravanti;Dilworth, F. Jeffrey
染色质免疫沉淀（ChIP）通常用于检查特定基因组位置的组蛋白的表观遗传修饰。然而，组蛋白尾部的共价修饰可以作为染色质调节因子的对接位点。因此，这些调控因子与染色质的结合可能导致抗体识别的空间位阻，导致低估特定基因座处给定组蛋白修饰的相对富集。为了克服这个问题，我们已经开发了一个天然的ChIP协议来研究组蛋白的共价修饰，该协议利用羟基磷灰石（HAP）层析在核小体的免疫沉淀之前洗掉染色质相关蛋白。这种快速而简单的程序由五个步骤组成：从培养细胞中分离细胞核;使用MNase进行染色质片段化;使用HAP纯化核小体;修饰核小体的免疫沉淀;以及与修饰组蛋白相关的DNA的qPCR分析。以这种方式制备的核小体不含污染蛋白质，并且允许准确评估特定基因组基因座处不同共价组蛋白修饰的相对丰度。完成该方案需要大约1.5天。
Chromatin immunoprecipitation (ChIP) is routinely used to examine epigenetic modification of histones at specific genomic locations. However, covalent modifications of histone tails can serve as docking sites for chromatin regulatory factors. As such, association of these regulatory factors with chromatin could cause steric hindrance for antibody recognition, resulting in an underestimation of the relative enrichment of a given histone modification at specific loci. To overcome this problem, we have developed a native ChIP protocol to study covalent modification of histones that takes advantage of hydroxyapatite (HAP) chromatography to wash away chromatin-associated proteins before the immunoprecipitation of nucleosomes. This fast and simple procedure consists of five steps: nuclei isolation from cultured cells; fragmentation of chromatin using MNase; purification of nucleosomes using HAP; immunoprecipitation of modified nucleosomes; and qPCR analysis of DNA associated with modified histones. Nucleosomes prepared in this manner are free of contaminating proteins and permit an accurate evaluation of relative abundance of different covalent histone modifications at specific genomic loci. Completion of this protocol requires similar to 1.5 d.