Abstract
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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Acknowledgements
This research was originally published in The Journal of Biological Chemistry. Kamyar Hadian, Richard A. Griesbach, Scarlett Dornauer, Tim M. Wanger, Daniel Nagel, Moritz Metlitzky, Wolfgang Beisker, Marc Schmidt-Supprian, and Daniel Krappmann. NF-κB Essential Modulator (NEMO) Interaction with Linear and Lys-63 Ubiquitin Chains Contributes to NF-κB Activation. Journal of Biological Chemistry. 2011; 286:26107–26117. © The American Society for Biochemistry and Molecular Biology.
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Vincendeau, M., Krappmann, D., Hadian, K. (2015). In Vitro Detection of NEMO–Ubiquitin Binding Using DELFIA and Microscale Thermophoresis Assays. In: May, M. (eds) NF-kappa B. Methods in Molecular Biology, vol 1280. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2422-6_18
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DOI: https://doi.org/10.1007/978-1-4939-2422-6_18
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2421-9
Online ISBN: 978-1-4939-2422-6
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