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Method for the Purification of Endogenous Unanchored Polyubiquitin Chains

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Proteostasis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1449))

Abstract

Unanchored polyubiquitin chains are endogenous non-substrate linked ubiquitin polymers which have emerging roles in the control of cellular physiology. We describe an affinity purification method based on an isolated ubiquitin-binding domain, the ZnF_UBP domain of the deubiquitinating enzyme USP5, which permits the selective purification of mixtures of endogenous unanchored polyubiquitin chains that are amenable to downstream molecular analyses. Further, we present methods for detection of unanchored polyubiquitin chains in purified fractions.

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Author information

Authors and Affiliations

  1. School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK

    Daniel Scott, Varun Gopala Krishna, Barry Shaw, David J. Tooth & Rob Layfield

  2. Institute of Cell Biology, University of Edinburgh, Edinburgh, UK

    Jo Strachan

  3. School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK

    Mark S. Searle & Neil J. Oldham

  4. Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK

    Mark S. Searle

Authors
  1. Daniel Scott
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  2. Jo Strachan
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  3. Varun Gopala Krishna
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  4. Barry Shaw
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  5. David J. Tooth
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  6. Mark S. Searle
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  7. Neil J. Oldham
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  8. Rob Layfield
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Corresponding author

Correspondence to Rob Layfield .

Editor information

Editors and Affiliations

  1. Computational and Experimental Biology Group Department of Health Promotion and Chronic Diseases, National Health Institute Dr. Ricardo Jorge, INSA, I.P., Lisboa, Portugal

    Rune Matthiesen

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Scott, D. et al. (2016). Method for the Purification of Endogenous Unanchored Polyubiquitin Chains. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_11

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  • DOI: https://doi.org/10.1007/978-1-4939-3756-1_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3754-7

  • Online ISBN: 978-1-4939-3756-1

  • eBook Packages: Springer Protocols

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