In vitro detection of NEMO-ubiquitin binding using DELFIA and microscale thermophoresis assays.
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.
Mesh Terms:
Fluorescent Antibody Technique, Gene Expression, I-kappa B Kinase, In Vitro Techniques, Protein Binding, Recombinant Fusion Proteins, Ubiquitin
Fluorescent Antibody Technique, Gene Expression, I-kappa B Kinase, In Vitro Techniques, Protein Binding, Recombinant Fusion Proteins, Ubiquitin
Methods Mol. Biol.
Date: Mar. 05, 2015
PubMed ID: 25736757
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