An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants

Abdelaty Saleh; Raúl Alvarez-Venegas; Zoya Avramova

doi:10.1038/nprot.2008.66

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants

Abdelaty Saleh, Raúl Alvarez-Venegas, Zoya Avramova

Research output: Contribution to journal › Article › peer-review

507 Scopus citations

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 μg 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.

Original language	English (US)
Pages (from-to)	1018-1025
Number of pages	8
Journal	Nature protocols
Volume	3
Issue number	6
DOIs	https://doi.org/10.1038/nprot.2008.66
State	Published - May 2008
Externally published	Yes

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

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10.1038/nprot.2008.66

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title = "An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants",

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 μg 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.",

author = "Abdelaty Saleh and Ra{\'u}l Alvarez-Venegas and Zoya Avramova",

note = "Funding Information: ACKNOWLEDGMENTS The authors are grateful to Dr Malali Gowda for critically reading the manuscript and Dr Justin Goodrich for his gift of clf mutant seeds. This work was partially supported by the NSF grant MCB-0343934 to Z.A.",

year = "2008",

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AU - Avramova, Zoya

N1 - Funding Information: ACKNOWLEDGMENTS The authors are grateful to Dr Malali Gowda for critically reading the manuscript and Dr Justin Goodrich for his gift of clf mutant seeds. This work was partially supported by the NSF grant MCB-0343934 to Z.A.

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N2 - 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 μg 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.

AB - 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 μg 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.

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An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants

Abstract

ASJC Scopus subject areas

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