Chromatin Immunoprecipitation in Human and Yeast Cells.
Chromatin Immunoprecipitation in Human and Yeast Cells.
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人类和酵母细胞中的染色质免疫沉淀。
DOI：
10.1007/978-1-4939-7774-1_14
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Keung,AlbertJ
中科院分区：
文献类型：
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作者：
Lee,JessicaB;Keung,AlbertJ
染色质免疫沉淀(ChIP)是研究蛋白质与基因组DNA相互作用的重要方法，如转录因子的基因组定位和组蛋白的翻译后修饰。DNA和蛋白质使用甲醛进行可逆的共价交联。然后裂解细胞以释放染色质。通过微球菌核酸酶(MNase)或超声波作用，染色质被分割成较小的尺寸，然后从其他细胞成分中纯化。蛋白质-DNA复合体通过免疫沉淀(IP)与针对感兴趣表位的抗体进行浓缩。通过加热和蛋白酶处理从蛋白质中释放DNA，然后用RNase降解受污染的RNA。使用各种方法分析得到的DNA，包括聚合酶链式反应、定量聚合酶链式反应或测序。这份协议概述了酵母和人类细胞的每一个步骤。
Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and posttranslational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MNase) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including PCR, qPCR, or sequencing. This protocol outlines each of these steps for both yeast and human cells.