| Assay Method Information | |
| | In vitro VEGFR Kinase Inhibition Assay |
| Description: | The inhibition was carried out in 96-well polystyrene round bottomed plates. These plates were precoated with 100 ul per well of 50 ug per ml poly(Glu:Tyr, 4:1) peptide (Sigma) in PBS. The kinase reaction was performed in the plates by addition of 50 ul of kinase buffer (50 mM HEPES, 125 mM NaCl, 10 mM MgCl2, pH 7.4) containing 100 uM of ATP, 10 ng of KDR (Invitrogen, catalytic domain of VEGFR2), and the compound to be tested (0.1 uM, 0.5 uM, 1 uM, 5 uM and 10 uM). The compounds were dissolved in DMSO and were further diluted with PBS. After 30 min of exposure, the plates were washed 2x3 times with PBS and inoculated with 50 ul per well of 0.2 ug per ml HRP conjugated antiphosphotyrosine antibody (Santa Cruz). After two washes, the plates were developed by addition of 50 ul per well tetramethylbenzidine (Sigma) and the reaction was terminated by addition of 50 ul per well of 2 N H2SO4. The absorbance at 450 nm was measured by a 96-well plate reader (Tecan) [Bioorg. Med. Chem. Lett., 16 (2006) 5127-5131. |
| Affinity data for this assay | |
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