| Assay Method Information | |
| | VEGFR2 (KDR) Enzyme Assayy |
| Description: | VEGFR2 (100 nM typically) was activated at room temperature for 20 min. in the presence of 100 mM HEPES, pH 7.5, 10 mM MgCl2, 100 μM ATP, 300 μM DTT, and 0.1 mg/mL BSA. A substrate solution containing 20 mM MgCl2, 100 μM ATP, 0.72 μM biotinylated peptide (Biotin-aminohexyl-EEEEYFELVAKKKK-NH2), was added in 5 μL to the assay plate. The solution of activated VEGFR2 was diluted 100-fold in 200 mM HEPES, pH 7.5, 0.2 mg/mL BSA, and 0.6 mM DTT. The VEGFR2 catalyzed reaction was initiated by the addition of 5 μL of the diluted, activated VEGFR2. Final assay concentrations were 100 mM HEPES, pH 7.5, 10 mM MgCl2, 50 μM ATP, 0.1 mg/mL BSA, 300 μM DTT, 0.36 μM biotinylated peptide substrate, and 0.5 nM VEGFR2 (the final assay concentration of VEGFR2 may vary depending on the specific activity of different batches of enzyme). The reaction was run for 90 min. at room temperature and then terminated by the addition of 5 μL of 150 mM EDTA, pH 8. The background signal of the assay was established in wells where the addition of the 150 mM EDTA was instead made prior to adding substrate and enzyme solutions. HTRF detection solution containing 200 mM HEPES, pH 7.5, 0.1 mg/mL BSA, 30 nM Streptavidin SureLight®-APC (PerkinElmer, Boston, Mass., USA), and 4 nM LANCE® europium-labelled antiphosphotyrosine antibody (Perkin Elmer, Boston, Mass., USA) was added in 5 μL. After incubation for 10 min. at room temperature, phosphorylation of the biotinylated peptide substrate was measured as a ratio of specific 665 nm energy transfer signal to reference europium 615 nm signal using a Viewlux 1430 ultraHTS Microplate Imager (PerkinElmer, Turku, Finland). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |