Quantitative Evaluation of VEGF Binding to VEGFR2 in Real Time in Living Cells

Vascular endothelial growth factors (VEGF) are proteins that act as dimers to mediate angiogenesis by binding to their cellular receptors (VEGFR). Upon ligand binding VEGFRs dimerize and initiate a raft of signaling cascades via their intracellular tyrosine kinase domains. VEGFs and their receptors play a role in tumor vascularization and are major targets of anti-cancer therapies. In order to characterize affinities of VEGF isoforms binding to VEGFR2, assays are required that quantitatively and kinetically report on these interactions. A novel labeling technique stoichiometrically labels the VEGF family member VEGF165a with the fluorophore TMR. The modified ligand was used in conjunction with NanoLuciferase-coupled VEGFR2 (NLuc VEGFR2) in a cellular NanoBRET assay.