| Assay Method Information | |
| | VEGFR ADP GLO Assay |
| Description: | Assay Procedure:Step 1. Dispensing inhibitors/controls: Using Echo, dispense 10 nL/well compound serial dilutions in DMSO to columns 1-22 (in 384-well plates) or columns 1-44 (in 1536-well plates). Dilution series=11 pt, 3-fold dilutions. The top compound concentration in the source plate is 4 mM. The top compound concentration in the assay plate is 10 uM. Using Echo, dispense 10 nl/well DMSO to column 23 (in 384-well plates) or columns 45-47 (in 1536-well plates). These wells will serve as negative control wells Using Echo, dispense 10 nl/well 400 uM TAK-593 in DMSO to column 24 (in 384-well plates) or column 48 (in 1536-well plates). The final concentration of TAK-593 in the assay should be 1 uM. These wells will serve as positive control wells.Step 2. Pre-incubation of inhibitors with kinase: Add 2 μL/well 2× kinase solution. Centrifuge at 1000 rpm for 1 min. Incubate at room temperature for 30 min.Step 3. Kinase cascade reaction: Add 2 μL/well 2× substrate/ATP solution to initiate kinase reactions. Centrifuge at 1000 rpm for 1 min. Incubate at room temperature for 180 min.Final concentrations of components in the assay:[VEGFR2]=5 nM;[ATP]=1.2 mM;[Srctide]=1 mg/mL;[DMSO] G; 1%.Step 4. Quench: Add 2 uL/well ADP Glo Reagent+0.05% CHAPS. Centrifuge at 1000 rpm for 1 min; Incubate at room temperature for one hour.Step 5. Detection: Add 2 uL/well Kinase Detection Reagent+0.05% CHAPS. Centrifuge at 1000 rpm for 1 min; Incubate at room temperature for 1 hour; Read Luminescence on a plate reader. |
| Affinity data for this assay | |
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