Chromatin immunoprecipitation and microarray-based analysis of protein location
Chromatin immunoprecipitation and microarray-based analysis of protein location
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DOI：
10.1038/nprot.2006.98
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Young, Richard A.
中科院分区：
文献类型：
--
作者：
Lee, Tong Ihn;Johnstone, Sarah E.;Young, Richard A.
全基因组定位分析，也称为芯片染色质免疫沉淀（ChIP - Chip），将染色质免疫沉淀和DNA微阵列分析相结合，以鉴定在活细胞中发生的蛋白质 - DNA相互作用。蛋白质 - DNA相互作用通过化学交联在体内被捕获。细胞裂解、DNA片段化以及对所需蛋白质进行免疫亲和纯化将共同纯化与该蛋白质相关的DNA片段。然后对富集的DNA群体进行标记，与差异标记的参照样本混合，并应用于DNA微阵列以检测富集信号。接着应用各种计算和生物信息学方法对富集通道和参照通道进行归一化，将信号与DNA微阵列上所代表的基因组部分相关联，提供置信度指标并生成蛋白质 - 基因组占据图谱。在此，我们描述了从细胞交联到标记物质杂交所使用的实验方案，以及对这些方案中影响结果的方面的见解。一旦获得足够数量的细胞，这些方案大约需要1周完成，并且已用于在许多不同的细胞和组织类型中产生可靠的、高质量的芯片染色质免疫沉淀结果。
Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types.