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

Nature Protocols volume 3, pages 1018–1025 (2008)Cite this article

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.

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Figure 1: Principal steps and duration of chromatin immunoprecipitation protocol.
Figure 2: Histone H3 Lys 27 methylation profiles of Flowering Locus T (FT) nucleosomes in clf mutant background.

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Acknowledgements

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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Authors and Affiliations

  1. School of Biological Sciences, University of Nebraska, Lincoln, 68588, Nebraska, USA

    Abdelaty Saleh, Raúl Alvarez-Venegas & Zoya Avramova

  2. Department of Plant Pathology, Center for Integrated Fungal Research (CIFR), North Carolina State University, Raleigh, 27695, North Carolina, USA

    Abdelaty Saleh

  3. Department of Genetic Engineering, Centro de Investigación y de Estudios Avanzados, Campus-Guanajuato, Irapuato, C.P. 36821, México

    Raúl Alvarez-Venegas

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  1. Abdelaty Saleh
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  3. Zoya Avramova
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Correspondence to Abdelaty Saleh or Zoya Avramova.

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Saleh, A., Alvarez-Venegas, R. & Avramova, Z. An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 3, 1018–1025 (2008). https://doi.org/10.1038/nprot.2008.66

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