| Assay Method Information | |
| | TR-FRET Assay |
| Description: | Compound Preparation. Dissslove the compounds into DMSO solution to make 10 mM DMSO stock. Serially dilute the stock solution from highest concentration down to lowest in DMSO to make the compound stock plate (100× stock plates). To make 5× compound intermediate plate, add 38 μL of assay buffer (50 mM HEPES, pH 7.5, 0.05 mg/mL BSA, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 2 mM DTT) into each well of the V-bottom plate; then transfer 2 μL of the stock compound solution of each concentration from the compound stock plate (100× stock), and mix well. Enzyme reaction. Transfer 2 μL compound from 5× intermediate plate into assay plate, then add 4 μL/well 2.5× Enzyme mix to assay plate, incubation for 10 min at room temperature. For positive control wells, add 4 μL/well assay buffer instead of Enzyme mix. Then add 4 μL/well 2.5× Substrate mix to assay plate, and incubation at 25° C. for 30 min at room temperature. After reaction, 10 μL 2× Detection mix (final 15 mM EDTA, 1× LANCE Detection Buffer, 0.5 nM Detection antibody) was added and incubated for 1 h prior to detection with excitation and emission at 320 and 665 nm (Tecan Spark® TR-FRET mode Tecan). Detail information of enzyme, substrate, detection antibody is listed in the following table. |
| Affinity data for this assay | |
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