染色质免疫沉淀剖析手艺效劳
一、手艺简介
染色质免疫沉淀法（Chromatin immunoprecitation ，ChIP）是研究体内DNA与卵白质相互作用的主要工具 。它可以迅速地检测目的卵白与特异DNA片断的连系情形 ，还可以用来研究组卵白与基因表达的关系 。它能真实、完整地反应连系在DNA序列上的调控卵白 ，是现在确定与特定卵白连系的基因组区域或确定与特定基因组区域连系的卵白质的最好要领 。
基来源理是在活细胞状态下牢靠卵白质－DNA复合物 ，并将其随机切断为一定长度规模内的染色质小片断 ，然后通过免疫学要领沉淀此复合体 ，特异性地富集目的卵白连系的DNA片断 ，通过对目的片断的纯化与检测 ，从而获得卵白质与DNA相互作用的信息 。它能真实、完整地反应连系在DNA序列上的调控卵白 ，是现在确定与特定卵白连系的基因组区域或确定与特定基因组区域连系的卵白质的一种很好的要领 。
二、实验办法
1、 甲醛交联
2、 超声打碎DNA
3、 免疫沉淀
4、 消化卵白质及解交联
5、 PCR扩增目的启动子片断
三、应用实例
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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