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

Nature Protocols volume 3, pages 10181025 (2008) | Download Citation

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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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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.

Author information

Affiliations

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

    • Abdelaty Saleh
    • , Raúl Alvarez-Venegas
    •  & Zoya Avramova
  2. Center for Integrated Fungal Research (CIFR), Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695, 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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Correspondence to Abdelaty Saleh or Zoya Avramova.

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DOI

https://doi.org/10.1038/nprot.2008.66

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