| Assay Method Information | |
| | VEGFR-2 Tyrosine Kinase Activity Assay |
| Description: | Kinase assays were performed in 96-well plates (Multiscreen Durapore, Millipore) using [γ-32P]ATP (Amersham Biosciences) and the synthetic polymer poly(Glu4/Tyr)(Sigma Chemicals) as a phosphoacceptor substrate. Tested compounds were dissolved in 1H-NMR DMSO, and the final concentration of 1H-NMR DMSO in assay solutions was0.1% (total kinase activity) or 0.01% (EGFR kinase activity), which was shown to have no effect on kinase activity. 10 ng of VEGFR-2 (recombinant human protein; Invitrogen) was incubated for 1 h at 28°C using various concentrations of tested compounds in kinase buffer (Tris 50mM pH 7.5, BSA 25 μg/mL, MnCl2 1.5 mM, MgCl2 10 mM, DTT 2.5 mM, Na3VO4 100 μM, β-glycerophosphate 5 mM, poly(Glu4/Tyr) 250 μg/mL, ATP 5 μM, [γ-32P]ATP 0.5 μCi). |
| Affinity data for this assay | |
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