| Assay Method Information | |
| | Inhibition of PARG Enzymatic Assay (TR-FRET) |
| Description: | PARG enzyme was incubated with compound or vehicle (DMSO) and the biotinylated-PARylated PARP-1 substrate in a microtiter plate. After adding detection antibody and streptavidin-europium, and then incubating, the plate was read for fluorescence intensity. The low control (DMSO) with low fluorescence intensity represents no inhibition of enzymatic activity, while the high control (no enzyme) with high fluorescence intensity represents full inhibition of enzymatic activity.Materials:Enzyme:PARGhPARG: 250 pM, 1-976, His-tagged, Proteos, 2.0 mg/mL (17.9 μM)Substrate: 30 nMTest Compound/Enzyme Pre-incubation time: 1 hrEnzyme/Substrate Reaction time: 10 minutesSubstrate: hPARP1, His6-TEV tagged, 1.2 mg/mL (10.3 μM)Detection Antibody: anti-His monoclonal antibody-ULight, Perkin Elmer catalog #TRF0134-MStreptavidin-Europium: Perkin Elmer catalog #AD0062Assay Buffer: 50 mM Tris-HCL pH 7.4, 50 mM KCL, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 0.01% BSATemperature: 23° C.Total reaction volume: 20 μLControls:0% inhibition: DMSO100% inhibition: No enzymeEnzyme reaction and Detection:1. Transfer 200 nL of 100× compound or DMSO to the appropriate wells of a 384 well white polystyrene microtitre plate (Corning Catalog #3574).2. Transfer 10 uL of 2× final concentration of enzyme in assay buffer or assay buffer alone to the appropriate wells.3. Centrifuge the plate at 1000 rpm for 30 seconds.4. Incubate the plate at room temperature for 1 hour.5. Transfer 10 uL of 2× substrate in assay buffer to all test wells.6. Incubate the plate at room temperature for 10 minutes7. Transfer 10 uL of 3× mixture of 42 nM detection antibody and 2.25 nM streptavidin-europium in 50 mM Tris-HCL pH 7.4 to all test wells.8. Incubate the plate at room temperature for 1 hour.9. Read the plate on a plate reader (Envision)Excitation: 317 nMEmission: 620 nMEmission: 665 nM |
| Affinity data for this assay | |
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