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. 2016:1449:203-13.
doi: 10.1007/978-1-4939-3756-1_11.

Method for the Purification of Endogenous Unanchored Polyubiquitin Chains

Daniel Scott  1 , Jo Strachan  2 , Varun Gopala Krishna  1 , Barry Shaw  1 , David J Tooth  1 , Mark S Searle  3   4 , Neil J Oldham  3 , Rob Layfield  5
Affiliations

Method for the Purification of Endogenous Unanchored Polyubiquitin Chains

Daniel Scott et al. Methods Mol Biol. 2016.

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.

Keywords: Free polyubiquitin; USP5; Ubiquitin; Ubiquitin-binding domain; Unanchored polyubiquitin; ZnF_UBP domain.

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