Contact EN Microplate reader Menu BMG LABTECHResourcesApplication notesAN317 317 NanoBRET™ assay quantitatively evaluates VEGF binding to the VEGFR2 in real-time in living cells Kilpatrick LE (1) Friedman-Ohana R (2) Alcobia DC (1) Riching K (2) Peach CJ (1) Wheal AJ (1) Briddon SJ (1) Robers MB (2) Zimmerman K (2) Machleidt T (2) Wood KV (2) Woolard J (1) Hill SJ (1) (1) Division of Physiology, Pharmacology and Neuroscience, University of Nottingham, UK (2) Promega Corporation, Madison, WI, USA 01/2018 Download application note PDF Novel labelling technique ﬂuorescently labels VEGF for use in NanoBRET interaction studies Assay acquires quantitative information on ligand-receptor interaction PHERAstar® simultaneously detects luciferase and ﬂuorophore signal for accelerated and kinetic reads Table of contents {{ item.content }} Introduction 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. Also interesting for you Learn which features help to easily resolve your binding events on a microplate reader. Read more Assay Principle VEGF165a was expressed as a HaloTag fusion protein that upon secretion was captured on Halolink resin. The tag was proteolytically removed by HaloTEV and in the presence of 6-TMR-PEG-CBT resulting in the ligand labelled with TMR at a single N-terminal cysteine (Fig. 1A) Fig. 1: Synthesis of VEGF165a (A) and the principle of the NanoBRET assay used to study VEGF-TMR binding to NLuc-VEGFR2 (B). Data taken from reference 1. The binding of the VEGF165a-TMR homodimer to NLuc fused VEGFR2 was detected using BRET. Brieﬂy, oxidation of the NLuc substrate furimazine produces energy which can be transferred from the bioluminescent donor (NLuc) to the acceptor ﬂurophore (TMR) if the two proteins are in sufﬁciently close proximity (<10nm). Hence, the ratio of ﬂuorophore to luciferase emission indicates interaction of ﬂuorescent ligand with its cognate receptor (Fig 1B). Materials & Methods White ﬂat bottomed microplates (Greiner Bio-One #655098) PHERAstar® FS Furimazine (N1130, Promega Corporation) HEK293 stably expressing NLuc-VEGFR2 Experimental procedure HEK293 cells expressing NLuc-VEGFR2 (40,000/well) were seeded 24 h prior to the experiment. Measurements were conducted in HBSS/0.1 % protease free BSA and with a furimazine concentration of 10 µM. Fluorescently labelled VEGF165-TMR, unlabelled VEGF165a, and Cediranib (an RTK inhibitor, 1 µM) were used at indicated concentrations and measurements were acquired with a PHERAstar FS microplate reader using the settings outlined below. Instrument settings Optic settings Luminescence, endpoint or plate mode kinetic, top optic Optic module NanoBRET; Em 1 460-80 (Nluc) Em 2 LP610 (TMR) Gain 2800 (Nluc) 3600 (TMR) General settings Settling time 0.0 s Measurement interval 1 s Kinetic settings Number of cycles 33 Cycle time 30 s Incubation Endpoint None Kinetic 37 °C Results & Discussion The TMR-labelled VEGF165a was added in concentrations from 1-20 nM to NLuc-VEGFR2 expressing HEK293 cells to monitor ligand-receptor interaction by NanoBRET. The time course of BRET ratios obtained shown in Figure 2 reveals the increase in binding and equilibrium of binding 10 min after the start of the reaction. Fig. 2: Ligand binding kinetics of VEGF165a-TMR to NLuc-VEGFR2 in HEK293 cells. Furimazine was added to each well and after 5 min of equilibration the reaction was started by adding VEGF165a-TMR at the indicated concentration (in nM). Data taken from reference 1. Next, the ability of the assay to determine ligand binding afﬁnities was tested by displacement of VEGF165a-TMR binding to VEGFR2 by unlabeled competing VEGF165a. The interaction between VEGF165a-TMR and VEGFR2 was impaired by increasing concentrations of the competing isoform as reported by loss of BRET signal (Fig. 3). The pKi for VEGF165a determined by BRET was 9.81. Fig. 3: Inhibition of VEGF165a-TMR binding to NLuc-VEGFR2 by VEGF165a. Fixed concentrations of VEGF165a-TMR (0.1–3 nM) were added simultaneously with increasing concentrations of the unlabeled VEGF165a and incubated for 1 h at 37 °C. Data represent mean ± S.E.M of 5 independent experiments, performed in triplicate. Bars represent the binding of VEGF165a-TMR obtained at each ﬂuorescent ligand concentration in the absence of competing ligand. Data taken from reference 1. The effect of the receptor tyrosine kinase inhibitor cediranib on VEGF165a-TMR binding to VEGFR2 was monitored for 2 h. In the absence of the inhibitor, ligand bound to VEGFR2 with a peak BRET signal obtained at approximately 15 min after ligand addition (Fig. 4, empty symbols). At later time points, the BRET signal reduced because of receptor internalization and subsequent complex dissociation. In contrast, in the presence of cediranib, high BRET ratios were observed that were sustained for the period of the experiment (2hr). This was due to cediranib preventing VEGFR2 internalisation, which was later conﬁrmed by confocal and super-resolution imaging (Kilpatrick et al., 2017 (1), Figure 7a; data not included in this report). Fig. 4: The effect of the receptor tyrosine kinase inhibitor cediranib on VEGF165a-TMR binding to VEGFR2 was monitored for 2 h. In the absence of the inhibitor, the ligand initially binds to VEGFR2 with a peak BRET signal obtained at approximately 15 min after ligand addition (Fig. 4, empty symbols). At later time points, the BRET signal reduces because of receptor internalization and subsequent complex dissociation. In contrast, presence of the RTKI cediranib prevents internalization and stabilizes the interaction for the period of the experiment (2 h). This becomes visible by sustained high BRET ratios (Fig. 4 full symbols). Data taken from reference 1. Conclusion The novel method for single site labeling a ligand with a ﬂuorophore has paved the way to use NanoBRET to quantify the molecular pharmacology of the anti-cancer target VEGFR2. The PHERAstar microplate reader reliably detected the luminescent and ﬂuorescent signals produced. Simultaneous acquisition of both signals accelerates the reading of a plate and increases the temporal resolution.The combination of up-to-date assay and detection technology helps to better understand and target cancer-related biological events. References 1. Kilpatrick LE et al. (2017) Biochem Pharmacol. Jul 15; 136:62-75. doi: 10.1016/j.bcp.2017.04.006 Lum AS FP TRF TR-FRET UV/vis FI Neph Back to Overview Compatible readers PHERAstar FSX Contact +49 781 96968-0 +49 781 96968-67 sales@bmglabtech.com Company Newsletter Privacy statement Legal notice EXTRAnet TeamViewer Support Legacy Plate Readers Luminometer Fluorometer Spectrophotometer Elisa plate reader High-throughput screening Neurodegenerative diseases Luminescence Social Media Copyright ©2021. All rights reserved.