| Assay Method Information | |
| | In Vitro c-Met and VEGFR-2 Enzyme Assay |
| Description: | Briefly, 20 μg/mL poly (Glu,Tyr) 4:1 (Sigma) was pre-coated in 96-well plates as a substrate. A 50 μL aliquot of 10 μmol/L ATP solution diluted in kinase reaction buffer (50 mmol/L HEPES [pH 7.4], 50 mmol/L MgCl2, 0.5 mmol/L MnCl2, 0.2 mmol/L Na3VO4, and 1 mmol/L DTT) was added to each well; 1 μL of various concentrations of indicated compounds diluted in 1% DMSO (v/v) (Sigma) was then added to each reaction well. DMSO (1%, v/v) was used as the negative control. The kinase reaction was initiated by the addition of purified tyrosine kinase proteins diluted in 49 μL of kinase reaction buffer. After incubation for 60 min at 37 °C, the plate was washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T-PBS). Anti-phosphotyrosine (PY99) antibody was then added. After a 30-min incubation at 37 °C, the plate was washed three times, and horseradishperoxidase-conjugated goat anti-mouse IgG was added. The plate was then incubated at 37 °C for 30 min and washed 3 times. A 100 μL aliquot of a solution containing 0.03% H2O2 and 2 mg/ml ophenylenediamine in 0.1 mol/L citrate buffer (pH 5.5) was added. The reaction was terminated by the addition of 50 μL of 2 mol/L H2SO4 as the color changed, and the plate was analyzed using a multi-well spectrophotometer (SpectraMAX 190, Molecular Devices) at 490 nm. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |