An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants
An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants
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DOI：
10.1038/nprot.2008.66
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Avramova, Zoya
中科院分区：
文献类型：
--
作者：
Saleh, Abdelaty;Alvarez-Venegas, Raul;Avramova, Zoya
染色质免疫沉淀（ChIP）技术是研究组蛋白共价修饰和DNA-组蛋白相互作用的有力工具。该程序包括染色质中的DNA-组蛋白交联，将DNA剪切成更小的片段，用针对感兴趣的组蛋白修饰的抗体进行免疫沉淀，然后通过PCR鉴定相关的DNA序列。在本协议中，我们描述了一个简化和优化版本的ChIP检测，减少了实验步骤和分离溶液的数量，缩短了制备时间。我们在染色质剪切之前包括核分离步骤，其提供高质量DNA的良好产量，从而从每个免疫沉淀样品（从0.2至0.4g起始组织材料）产生至少15 μ g DNA，足以测试>= 25个感兴趣的基因。这种简单和成本效益的协议已被应用于各种拟南芥组织的组蛋白修饰的研究，并且很容易适应其他系统以及。
Chromatin immunoprecipitation (ChIP) is a powerful tool for the characterization of covalent histone modifications and DNA-histone interactions in vivo. The procedure includes DNA-histone cross-linking in chromatin, shearing DNA into smaller fragments, immunoprecipitation with antibodies against the histone modifications of interest, followed by PCR identification of associated DNA sequences. In this protocol, we describe a simplified and optimized version of ChIP assay by reducing the number of experimental steps and isolation solutions and shortening preparation times. We include a nuclear isolation step before chromatin shearing, which provides a good yield of high-quality DNA resulting in at least 15 mu g of DNA from each immunoprecipitated sample (from 0.2 to 0.4 g of starting tissue material) sufficient to test >= 25 genes of interest. This simpler and cost-efficient protocol has been applied for histone-modification studies of various Arabidopsis thaliana tissues and is easy to adapt for other systems as well.