| Assay Method Information | |
| | In Vitro Test of the Compound's Inhibitory Effect on VEGFR2 Kinase |
| Description: | In Vitro Test of the Compound's Inhibitory Effect on VEGFR2 Kinase Experimental Methods and Procedures: The ADP-GLO Kinase Assay is an analytical reaction in which a luminescent kinase is used to determine the adenosine diphosphate formed by the kinase; ADP is converted to ATP, i.e., an optical signal is generated by Ultra-Glo™ luciferase. The optical signal is positively correlated with ADP quantity and kinase activity. This assay is ideal for determining the activity of the compound, making it ideal for primary screening and kinase selectivity analysis. The ADP-Glo™ Kinase Assay can be used to monitor the activity of virtually any ADP-producing enzyme such as a kinase orATPase: 1) The enzyme, substrate, ATP and inhibitor were diluted in a kinase buffer; 2) 384-well plate: 1 μL of inhibitor or (5% dimethyl sulfoxide solution), 2 μL of KDR enzyme, 2 μL of matrix/ATP mixture; 3) the mixture was incubated at room temperature for 60 minutes; 4) 5 μL of ADP-GLO™ reagent was added; 5) the mixture was incubated for 40 minutes at room temperature; 6) 10 μL of kinase detection reagent was added; 7) the mixture was incubated at room temperature for 30 minutes; 8) the luminescence was recorded (integration time 0.5-1 sec). The data was shown in relative light units (rlu), which was directly related to the amount of ATP produced. There was correlation between the percent of the conversion from ATP to ADP for each kinase amount and the corresponding signal-to-background ratio. 9) Data analysis: (a) KDR enzyme was titrated with 50 μM of ATP, and the luminescent signal produced by KDR enzyme was shown; (b) staurosporine dose response was generated with 1.5 ng of KDR to determine the IC50 value of the inhibitor. |
| Affinity data for this assay | |
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