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    Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest)

    Hospenthal, Manuela and Mevissen, T.E.T and Komander, D. (2015) Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest). Nature Protocols 10 (2), pp. 349-361. ISSN 1754-2189.

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    Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro. UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins.


    Item Type: Article
    Keyword(s) / Subject(s): Analytical biochemistry, Deubiquitylating enzymes, Post-translational modifications, Ubiquitins
    School: Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences
    Depositing User: Administrator
    Date Deposited: 06 Sep 2016 15:03
    Last Modified: 02 Aug 2023 17:25


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