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染色质免疫沉淀分析技术服务

编辑:   上传日期:2017-06-27  阅读次数:

一、技术概况

染色质免疫沉淀法(Chromatin immunoprecitation,ChIP)是研究体内DNA与蛋白质相互作用的重要工具。它可以灵敏地检测目标蛋白与特异DNA片段的结合情况,还可以用来研究组蛋白与基因表达的关系。它能真实、完整地反映结合在DNA序列上的调控蛋白,是目前确定与特定蛋白结合的基因组区域或确定与特定基因组区域结合的蛋白质的最好方法。
基本原理是在活细胞状态下固定蛋白质-DNA复合物,并将其随机切断为一定长度范围内的染色质小片段,然后通过免疫学方法沉淀此复合体,特异性地富集目的蛋白结合的DNA片段,通过对目的片断的纯化与检测,从而获得蛋白质与DNA相互作用的信息。它能真实、完整地反映结合在DNA序列上的调控蛋白,是目前确定与特定蛋白结合的基因组区域或确定与特定基因组区域结合的蛋白质的一种很好的方法。
   

二、实验步骤

1、 甲醛交联
2、 超声打碎DNA
3、 免疫沉淀
4、 消化蛋白质及解交联
5、 PCR扩增目的启动子片段
 

三、应用实例

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Fig1: ChIP analysis to detect in vivo binding of c-Jun to the dp5 promoter.
CGNs maintained in 25 or 5 K medium for 4 h were treated with formaldehyde to cross-link endogenous proteins and DNA. Samples of sonicated and purified chromatin were immunoprecipitated with a rabbit c-Jun antibody or a normal rabbit IgG. A, chromatins were sonicated into fragments about 0.5 kb in length. B, Western blotting analysis (WB) was performed using a monoclonal c-Jun antibody to demonstrate the immunoprecipitation (IP) specificity and efficiency and analyze c-Jun cellular protein levels. C, left, DNA isolated and purified from immunoprecipitated material was amplified by PCR with primers to amplify a 169-bp fragment of dp5 promoter spanning the ATF site (top), and a 173-bp fragment encompassing a canonical ATF sequence (TRE-jun2 site) located in the c-jun promoter was also amplified (bottom). The amplified PCR fragments were analyzed on 2% agarose gel.C, right, equal amounts of total genomic DNA (Input) were used for immunoprecipitation in each condition. Data are representative of three separate experiments.

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