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.
Research Unit Cellular Signal Integration, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
Michelle
M
Vincendeau
Daniel
D
Krappmann
Kamyar
K
Hadian
D005455:Fluorescent Antibody Technique / Q000379:methods*; D015870:Gene Expression; D051550:I-kappa B Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D051550:I-kappa B Kinase