A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells
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Forment, J., & Jackson, S. (2015). A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells. Nature Protocols, 10 1297-1307. https://doi.org/10.1038/nprot.2015.066
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, make it easier to quantify and allow a stream-lined application in high‐throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry 1. 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 (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy.
flow cytometry, chromatin, chromatin association, chromatin‐bound, immunofluorescence microscopy
Research in our laboratory is 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 is provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). J.V.F. is funded by Cancer Research UK programme grant C6/A11224 and the Ataxia Telangiectasia Society. S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK.
Cancer Research UK (11224)
Cancer Research UK (A14492)
Wellcome Trust (092096/Z/10/Z)
European Research Council (268536)
External DOI: https://doi.org/10.1038/nprot.2015.066
This record's URL: https://www.repository.cam.ac.uk/handle/1810/248571