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Sequential Chromatin Immunoprecipitation Assay and Analysis

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Epigenetics Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 791))

Abstract

Sequential chromatin immunoprecipitation (SeqChIP) assays have been developed for the study of interactions of two or more proteins (or simultaneous histone modifications) at genomic sites. It is based on the principle that chromatin and associated proteins can be first immunoprecipitated with a first antibody and the obtained immunoprecipitate can be subjected to a second antibody. At the end of the assay the immunoprecipitated material contains only chromatin that concomitantly carries both DNA-associated proteins (or both histone modifications). The SeqChIP protocol described here combines speed (minimum of 3–4 h to perform the complete assay), sensitivity (known targets can be detected with only about 20,000 cell equivalents), and avoidance of antibody–antigen disruption after the first ChIP step. In addition, specific SeqChIP controls and potential shortcomings are discussed, the main characteristics of different SeqChIP protocols are described and several examples of protein complexes and protein–protein interactions at genomic sites that have been solved by SeqChIP in the recent years are presented.

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Acknowledgments

The author would like to thank colleagues at R&D Systems who contributed for the development of this protocol, including Ernesto Resnik, Brian Hoium, and Jessie Ni.

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Authors and Affiliations

  1. Department of Antibody Applications and Stem Cells, R&D Systems, Inc., Minneapolis, MN, USA

    Ricardo B. de Medeiros

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  1. Ricardo B. de Medeiros
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Correspondence to Ricardo B. de Medeiros .

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Editors and Affiliations

  1. Dept. Biology, University of Alabama, Birmingham, University Blvd. 1300, Birmingham, 35294-1170, Alabama, USA

    Trygve O. Tollefsbol

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de Medeiros, R.B. (2011). Sequential Chromatin Immunoprecipitation Assay and Analysis. In: Tollefsbol, T. (eds) Epigenetics Protocols. Methods in Molecular Biology, vol 791. Humana Press. https://doi.org/10.1007/978-1-61779-316-5_17

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  • DOI: https://doi.org/10.1007/978-1-61779-316-5_17

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-315-8

  • Online ISBN: 978-1-61779-316-5

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