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NanoBRET™ assay quantitatively evaluates VEGF binding to the VEGFR2 in real-time in living cells

Kilpatrick LE1, Friedman-Ohana R2, Alcobia DC1, Riching K2, Peach CJ1, Wheal AJ1, Briddon SJ1, Robers MB2, Zimmerman K2, Machleidt T2,, Wood KV2, Woolard J1, Hill SJ1 1Division of Physiology, Pharmacology and Neuroscience, University of Nottingham, UK, 2Promega Corporation, Madison, WI, USA 01/2018

Receptor tyrosine kinases (RTK) are transmembrane receptors that translate stimulation by growth factors into regulation of cell growth, proliferation, cell death and differentiation. Consequently, aberrant RTK-signaling is implicated in cancer, making it a popular anti-cancer drug target. Development of pharmaceutical RTK-inhibitors requires assays that characterize receptor-ligand interaction and that identify inhibitors thereof.


A novel method labeled the vascular endothelial growth factor (VEGF) at a single site with the fluorophore TMR. The labeled ligand was used in combination with HEK293 cells expressing the RTK VEGF-receptor 2 (VEGFR2) fused to the NanoLuc® luciferase. Upon interaction of ligand and receptor, bioluminescence resonance energy transfer (BRET) between luciferase and fluorophore takes place.


The signal of luciferase and fluorophore were acquired simultaneously with the PHERAstar microplate reader. This way, not only the pKi of a VEGF-inhibitor was determined, but also the time-course of binding and inhibition was monitored.

 

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