一、技術(shù)簡介

染色質(zhì)免疫沉淀法（Chromatin immunoprecitation，ChIP）是研究體內(nèi)DNA與蛋白質(zhì)相互作用的重要工具。它可以靈敏地檢測目標(biāo)蛋白與特異DNA片段的結(jié)合情況，還可以用來研究組蛋白與基因表達(dá)的關(guān)系。它能真實(shí)、完整地反映結(jié)合在DNA序列上的調(diào)控蛋白，是目前確定與特定蛋白結(jié)合的基因組區(qū)域或確定與特定基因組區(qū)域結(jié)合的蛋白質(zhì)的最好方法。
基本原理是在活細(xì)胞狀態(tài)下固定蛋白質(zhì)－DNA復(fù)合物，并將其隨機(jī)切斷為一定長度范圍內(nèi)的染色質(zhì)小片段，然后通過免疫學(xué)方法沉淀此復(fù)合體，特異性地富集目的蛋白結(jié)合的DNA片段，通過對目的片斷的純化與檢測，從而獲得蛋白質(zhì)與DNA相互作用的信息。它能真實(shí)、完整地反映結(jié)合在DNA序列上的調(diào)控蛋白，是目前確定與特定蛋白結(jié)合的基因組區(qū)域或確定與特定基因組區(qū)域結(jié)合的蛋白質(zhì)的一種很好的方法。
   

二、實(shí)驗(yàn)步驟

1、 甲醛交聯(lián)
2、 超聲打碎DNA
3、 免疫沉淀
4、 消化蛋白質(zhì)及解交聯(lián)
5、 PCR擴(kuò)增目的啟動(dòng)子片段
 

三、應(yīng)用實(shí)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.