Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo.
Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo.
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DOI：
10.1002/0471142727.mb2103s69
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发表时间：
2005-02-01
影响因子：
--
通讯作者：
Struhl, Kevin
中科院分区：
文献类型：
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作者：
Aparicio, Oscar;Geisberg, Joseph V;Struhl, Kevin
染色质免疫沉淀（ChIP）是一种强大且广泛应用的技术，用于在体内检测单个蛋白质与特定基因组区域的关联。用甲醛处理活细胞，以使在体内染色质模板上紧密相邻的分子之间产生蛋白质 - 蛋白质和蛋白质 - DNA交联。与特定蛋白质交联的DNA序列被选择性富集，甲醛交联的逆转使得能够对免疫沉淀的DNA进行回收和定量分析。由于甲醛在加入细胞后基本上立即使细胞酶失活，ChIP提供了在特定时间点的蛋白质 - 蛋白质和蛋白质 - DNA相互作用的快照，因此对于体内染色体序列上发生的事件的动力学分析是有用的。此外，ChIP可与微阵列技术相结合，在全基因组基础上鉴定特定蛋白质的位置。本单元介绍了酿酒酵母的ChIP程序；描述了哺乳动物细胞的相应步骤。
Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein-protein and protein-DNA cross-links between molecules that are in close proximity on the chromatin template in vivo. DNA sequences that cross-link with a given protein are selectively enriched, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein-protein and protein-DNA interactions at a particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome-wide basis. in this unit describes the ChIP procedure for Saccharomyces cerevisiae; describes the corresponding steps for mammalian cells.