Protocol abstract
Nature Protocols 3, - 1018 - 1025 (2008)
Published online: 22 May 2008 | doi:10.1038/nprot.2008.66
Subject Categories: Biochemistry and protein analysis | Immunological techniques | Model organisms | Plant biology
An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants
Abdelaty Saleh1,2, Raúl Alvarez-Venegas1,3 & Zoya Avramova1
Abstract
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 
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.
- School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588, USA.
- Center for Integrated Fungal Research (CIFR), Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695, USA.
- Department of Genetic Engineering, Centro de Investigación y de Estudios Avanzados, Campus-Guanajuato, Irapuato, C.P. 36821, México.
Correspondence to: Abdelaty Saleh1,2 e-mail: asaleh@ncsu.edu
Correspondence to: Zoya Avramova1 e-mail: zavramova2@unl.edu
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A-Z product listing
- ACTIN7 forward 5'-GGTGAGGATATTCAGCCACTTGTCTG-3'(Integrated DNA Technologies)
- ACTIN7 reverse 5'-TGTGAGATCCCGACCCGCAAGATC-3'(Integrated DNA Technologies)
- Anti-dimethyl-histone H3(Upstate)
- Anti-trimethyl-histone H3(Upstate)
- Calcium chloride(Sigma)
- Cheesecloth miniwipes(Fisher)
