| Assay Method Information | |
| | Enzymatic Activity of hVEGFR2 |
| Description: | The enzymatic activity of hVEGFR2 was measured using the TR-FRET based Ulight assay (Revvity) which measures phosphorylation of a fluorescently tagged peptide substrate with the Europium labeled PT66 Antibody that specifically recognizes phosphorylated tyrosine residues. The assay is performed in a buffered solution (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 0.5 mM TCEP) with an ATP concentration of the measured Km of the VEGFR2 (150 M ATP). Enzyme concentrations of 50 pM VEGFR2 and Ulight peptide concentration of 50 nM Ulight-JAK1 were used. Reactions went 1 hour before being quenched with a quenching solution (buffered solution above with 20 mM EDTA and 0.5 nM Europium-anti-phosphotyrosine (PT66) Ab). The assay was further incubated for 30 minutes to allow for complete binding of the antibody and then read on a Envision plate reader to measure TR-FRET signal.DMSO solutions of potential TKI compounds were dispensed using an ECHO 655 to dispense into White Proxi-plus 384-well plates with a total DMSO volume of 50 nl. Each compound was assessed at 10 concentrations in 3-fold serial dilutions starting at a high concentration of 2.5 M and with a low concentration of 124 pM. After compound dispensing, 2.5 μl of a 4× concentration of ATP was added to each plate well. Then 2.5 μl if a 4× concentration of the tyrosine kinase was added to each well and allowed to incubate at RT for 30 minutes. After the incubation, the kinase reaction was started with the addition of 5 μl of a 2× concentration of one of the Ulight peptides and incubated at RT for 30 or 60 minutes as indicated above. The reaction was then quenched with the addition of 5 μl of the quenching solution. |
| Affinity data for this assay | |
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