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Vincendeau, M. ; Krappmann, D. ; Hadian, K.

In vitro detection of NEMO-ubiquitin binding using DELFIA and microscale thermophoresis assays.

Methods Mol. Biol. 1280, 311-320 (2015)
DOI PMC
Open Access Green as soon as Postprint is submitted to ZB.
Canonical NF-κB signaling in response to various stimuli converges at the level of the IκB kinase (IKK) complex to ultimately activate NF-κB. To achieve this, the IKK complex uses one of its regulatory subunit (IKKγ/NEMO) to sense ubiquitin chains formed by upstream complexes. Various studies have shown that different Ubiquitin chains are involved in the binding of NEMO and thereby the activation of NF-κB. We have utilized two distinct biochemical methods, i.e., Dissociation-Enhanced Lanthanide Fluorescence Immunoassay (DELFIA) and Microscale Thermophoresis (MST), to detect the interaction of NEMO to linear and K63-linked Ubiquitin chains, respectively. Here, we describe the brief basis of the methods and a detailed underlying protocol.
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Publication type Article: Journal article
Document type Scientific Article
Editors May, M.J.*
ISSN (print) / ISBN 1064-3745
e-ISSN 1940-6029
Conference Title NF-kappa B : Methods and Protocols
Journal Methods in Molecular Biology
Quellenangaben Volume: 1280, Issue: , Pages: 311-320 Article Number: , Supplement: ,
Publisher Springer
Publishing Place Berlin [u.a.]
Reviewing status Peer reviewed
Institute(s) Research Unit Signaling and Translation (SAT)
Institute of Molecular Toxicology and Pharmacology (TOX)