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A flow cytometry–based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

Nature Protocols volume 10, pages 1297–1307 (2015)Cite this article

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Abstract

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, to make it easier to quantify and to allow a streamlined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (takes no more than a working day from sample collection to quantification), requires less starting material compared with standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy.

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Figure 1: In situ extraction facilitates protein detection on chromatin.
Figure 2: Experimental workflow with some estimated timings.
Figure 3: Analysis of protein association to chromatin by flow cytometry.
Figure 4: Combining detection of protein association to chromatin with cell cycle markers.

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Acknowledgements

We thank all members of the Jackson laboratory for helpful discussions. We thank especially A. Blackford, G. Balmus, K. Dry, D.Weismann, C. Schmidt and P. Marco-Casanova for critical reading of the manuscript. Research in our laboratory was funded by Cancer Research UK (CRUK; programme grant C6/A11224), the European Research Council and the European Community Seventh Framework Programme (grant agreement no. HEALTH-F2-2010-259893 (DDResponse)). Core funding was provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). J.V.F. is funded by CRUK programme grant C6/A11224 and the Ataxia Telangiectasia Society. S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK.

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Authors and Affiliations

  1. The Wellcome Trust/Cancer Research UK (CRUK) Gurdon Institute, University of Cambridge, Cambridge, UK

    Josep V Forment & Stephen P Jackson

  2. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Josep V Forment & Stephen P Jackson

  3. The Wellcome Trust Sanger Institute, University of Cambridge, Cambridge, UK.,

    Josep V Forment & Stephen P Jackson

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  1. Josep V Forment
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J.V.F. conceived and developed the protocol. J.V.F. and S.P.J. wrote the manuscript.

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Correspondence to Josep V Forment.

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Forment, J., Jackson, S. A flow cytometry–based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells. Nat Protoc 10, 1297–1307 (2015). https://doi.org/10.1038/nprot.2015.066

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