| Assay Method Information | |
| | VEGFR2 Kinase Assay |
| Description: | VEGFR kinase assays were set up to assess the level of autophosphorylation based on DELFIA/Time-Resolved Fluorometry. 96-well plates were coated at room temperature for 1-2 h with 100 μL per well of 25 μg mL−1 poly-(Glu4-Tyr) peptide (Sigma) in Tris-buffered saline (TBS) (25 mM Tris, pH 7.2, 150 mM NaCl). Unbound peptide was washed three times with PBS. The cytoplasmic domain of VEGFR-2 enzyme was diluted (depending on the specific activity of the batch, from 10- to 20-fold) in 0.1% BSA/4 mM HEPES. A master mix of enzyme plus kinase buffer was prepared: (per well) 10 μL of diluted enzyme, 10 μL of kinase buffer (4 mM HEPES, pH 7.4, 1.25 mM MnCl2, 20 mM Na3VO4) and compounds (10 μL) prepared in 100% dimethyl sulfoxide (DMSO) were added to wells with appropriate density. Controls were done by adding DMSO alone, i.e., no test compound, to wells containing the master mix of enzyme plus kinase buffer. After 15 min at room temperature, ATP/MgCl2 (20 μL of |
| Affinity data for this assay | |
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