Category: Advances in Bioanalytics and Biomarkers
Cullin-RING E3 ubiquitin ligases (CRLs) are activated by neddylation and inactivated by de-neddylation that is accomplished by the COP9 signalosome (CSN). Trapping CRLs in their inactive state is reported to elevate tumor suppressors. Therefore, inhibition of CSN is a potential modality for cancer treatment.
Here, we show the development of a high-throughput microplate de-neddylation assay for a CRL substrate that was used to find inhibitors of CSN. A conventional fluorescence polarization approach was pursued based on the CSN5-mediated release of fluorophore-labelled NEDD8 from the CRL-substrate. Fluorescence polarization assays exploit the change in molecular weight of fluorophore-labeled molecules. The molecular weight influences the rotational speed of a molecule and consequently the polarization of emission light, the readout of fluorescence polarization assays. However, molecular weight difference from substrate-bound NEDD8 to free NEDD8 was not sufficient to provide an acceptable assay window for a reliable assay.
Tobias Pusterla– International Marketing Manager, BMG LABTECH GmbH, Ortenberg, Baden-Wurttemberg, Germany
International Marketing Manager
BMG LABTECH GmbH
Ortenberg, Baden-Wurttemberg, Germany
Tobias Pusterla is International Marketing Manager with BMG LABTECH. He graduated in Veterinary Biotechnology at the University of Milan, Italy. After an experience in the Core Facility for Mouse Mutagenesis in Milan, he obtained a Ph.D. in Cellular and Molecular Biology at the Open University of London, UK, in a joint program with the Hospital and Scientific Institute San Raffaele, Milan, Italy. Subsequently, he joined as a postdoc fellow the German Cancer Research Center (DKFZ) in Heidelberg, Germany. He joined BMG LABTECH in January 2013.