Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans
Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans
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DOI：
10.1038/nprot.2008.38
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Tissenbaum, Heidi A.
中科院分区：
文献类型：
--
作者：
Mukhopadhyay, Arnab;Deplancke, Bart;Tissenbaum, Heidi A.
为了确定信号通路如何差异调节基因表达，有必要确定转录因子（TF）和它们的同源顺式调控DNA元件之间的相互作用。在这里，我们已经概述了染色质免疫沉淀（ChIP）协议用于在整个秀丽隐杆线虫提取物。我们讨论了程序的优化，包括蠕虫的生长和收获，甲醛固定，TF免疫沉淀和通过实时PCR分析结合序列。获得用于ChIP的蠕虫培养物需要类似的10-12天; ChIP程序间隔2.5天，孵育两个过夜。
In order to determine how signaling pathways differentially regulate gene expression, it is necessary to identify the interactions between transcription factors (TFs) and their cognate cis-regulatory DNA elements. Here, we have outlined a chromatin immunoprecipitation (ChIP) protocol for use in whole Caenorhabditis elegans extracts. We discuss optimization of the procedure, including growth and harvesting of the worms, formaldehyde fixation, TF immunoprecipitation and analysis of bound sequences through real-time PCR. It takes similar to 10-12 d to obtain the worm culture for ChIP; the ChIP procedure is spaced out over a period of 2.5 d with two overnight incubations.