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The Nucleus pp 25–32Cite as

Isolation of Nuclei in Media Containing an Inert Polymer to Mimic the Crowded Cytoplasm

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1228))

Abstract

Within cells, the nucleus is surrounded by the cytoplasm which contains diffusible macromolecules at a high concentration (>100 mg/ml). When cells are broken to isolate nuclei by current methods these macromolecules are dispersed, and to reproduce the environment of nuclei in vivo more closely we have developed a method to isolate them in a medium where cytoplasmic macromolecules are replaced by an inert, volume-occupying polymer and which is essentially cation-free. Nuclei isolated by this method resemble closely those prepared by conventional procedures as seen by optical and electron microscopy, and their internal compartments (nucleoli, PML and Cajal bodies, transcription centers, and splicing speckles) and transcriptional activity are conserved. This procedure is efficient for mammalian cells that normally grow in suspension and do not have an extensive cytoskeleton, and requires ~30 min.

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Acknowledgements

We thank Jason Swedlow (Wellcome Trust Biocentre, University of Dundee, Scotland) for HeLa cells expressing GFP-coilin and David Bazett-Jones (Hospital for Sick Children, Toronto, Canada) for U2OS cells expressing GFP-PML isoform IV (originally from J. Taylor, Medical College of Wisconsin).

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Authors and Affiliations

  1. Laval University Cancer Research Centre-CRCHUQ Oncology, 9 rue MacMahon, Québec, QC, Canada, G1R2J6

    Ronald Hancock & Yasmina Hadj-Sahraoui

  2. Systems Biology Group, Biotechnology Centre, Silesian University of Technology, Gliwice, Poland

    Ronald Hancock

Authors
  1. Ronald Hancock
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  2. Yasmina Hadj-Sahraoui
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Corresponding author

Correspondence to Ronald Hancock .

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Editors and Affiliations

  1. Laval University Cancer Research Center, Québec, Québec, Canada

    Ronald Hancock

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Hancock, R., Hadj-Sahraoui, Y. (2015). Isolation of Nuclei in Media Containing an Inert Polymer to Mimic the Crowded Cytoplasm. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 1228. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1680-1_3

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  • DOI: https://doi.org/10.1007/978-1-4939-1680-1_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1679-5

  • Online ISBN: 978-1-4939-1680-1

  • eBook Packages: Springer Protocols

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