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TUBE and UbiCRest assays for elucidating polyubiquitin modifications in protein complexes
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).ORCID iD: 0000-0001-7622-5116
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).ORCID iD: 0000-0003-1170-0224
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).ORCID iD: 0000-0002-1269-8288
2019 (English)In: DNA SENSORS AND INFLAMMASOMES / [ed] Sohn, J, ELSEVIER ACADEMIC PRESS INC , 2019, p. 339-350Chapter in book (Refereed)
Abstract [en]

Ubiquitination is a reversible posttranslational modification that regulates nearly all cellular processes. The ubiquitin polypeptide is conjugated via its C-terminus to amine groups of lysine residues on target protein. Additionally, ubiquitins moieties can be conjugated in tandem to the initial ubiquitin via any of its internal lysine residues or N terminal methionine residue, resulting in the formation of polyubiquitin chains with distinct biophysical properties and biological functions. Elucidating the types of polyubiquitin chains present in proteins is essential for understanding their function and mechanism of regulation. Traditionally, ubiqutin modifications have been elucidated by exogenously co-expressing proteins of interest with epitope-tagged ubiquitins mutated in specific lysine residues. However, this strategy is prone experimental artifacts. In this protocol, we describe how to elucidate endogenous ubiquitin modifications. This procedure combines TUBE (Tandem Ubiquitin Binding Entity)-based isolation of ubiquitin conjugates, digestion with linkage specific deubiquitinases and immunoblotting. This procedure is very robust can be applied to profile types and architectural organization polyubiquitin chains present on the any proteins of interest and has been instrumental in elucidating ubiquitin modifications in NOD2 signaling in our recent study (Panda & Gekara, 2018).

Place, publisher, year, edition, pages
ELSEVIER ACADEMIC PRESS INC , 2019. p. 339-350
Series
Methods in Enzymology, ISSN 0076-6879 ; 625
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-164489DOI: 10.1016/bs.mie.2019.05.006ISI: 000488782900021PubMedID: 31455535ISBN: 978-0-12-818360-1 (electronic)ISBN: 978-0-12-818359-5 (print)OAI: oai:DiVA.org:umu-164489DiVA, id: diva2:1365627
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2019-10-25Bibliographically approved

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Panda, SwarupaJiang, HuiGekara, Nelson O.

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