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The Nucleus pp 15-23 | Cite as

Lysis Gradient Centrifugation: A Flexible Method for the Isolation of Nuclei from Primary Cells

  • Karl Katholnig
  • Marko Poglitsch
  • Markus Hengstschläger
  • Thomas WeichhartEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1228)

Abstract

The isolation of nuclei from eukaryotic cells is essential for studying the composition and the dynamic changes of the nuclear proteome to gain insight into the mechanisms of gene expression and cell signalling. Primary cells are particularly challenging for standard nuclear isolation protocols due to low protein content, sample degradation, or nuclear clumping. Here, we describe a rapid and flexible protocol for the isolation of clean and intact nuclei, which results in the recovery of 90–95 % highly pure nuclei. The method, called lysis gradient centrifugation (LGC), is based on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. A single low g-force centrifugation step enables mild cell lysis and prevents extensive contact of the nuclei with the cytoplasmic environment. This fast method shows high reproducibility due to the relatively little cell manipulation required by the investigator. Further advantages are the low amount of starting material required, easy parallel processing of multiple samples, and isolation of nuclei and cytoplasm at the same time from the same sample.

Key words

Nuclear isolation Mild lysis Fractionation Single step Discontinuous density gradient 

Notes

Acknowledgements

We would like to thank Werner Poglitsch for technical support in initial experiments.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Karl Katholnig
    • 1
  • Marko Poglitsch
    • 2
  • Markus Hengstschläger
    • 1
  • Thomas Weichhart
    • 1
    Email author
  1. 1.Institute for Medical GeneticsMedical University of ViennaViennaAustria
  2. 2.Attoquant Diagnostics GmbHViennaAustria

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