| Assay Method Information | |
| | Rho Kinase (ROCK) and Protein Kinase A (PKA) Enzyme Inhibition Assays |
| Description: | The assay is briefly described below.1. Diluted enzyme, substrate, ATP, and compounds (inhibitors) in Kinase Buffer (40 mM Tris, 7.5, 20 mM MgCl2, 0.1 mg/ml BSA, 50 μM DTT).2. Added 1 μl of inhibitor, 2 μl of enzyme and 2 μl of substrate/ATP mix to the wells of 384-well low volume nonbinding assay plate A series of three-fold dilution of inhibitor was prepared and assayed for each inhibitor.3. Incubated at room temperature for 60 minutes.4. Added 5 μl of ADP-Glo™ Reagent.5. Incubated at room temperature for 60 minutes.6. Added 10 μl of Kinase Detection Reagent7. Incubated at room temperature for 30 minutes.Recorded luminescence (Integration time 1 second) using a plate-based luminometer.Calculated Percent Enzyme Activity: First subtract the signal of the negative control (no enzyme and no inhibitor) from each sample's signal. Then use the mean relative light units (RLU) values for the 0% kinase activity (neither inhibitor nor enzyme) and the 100% kinase activity (no compound) to calculate the other percent enzyme activities remaining in the presence of the different dilutions of inhibitor.Plotted percent enzyme activity and inhibitor concentration dose response curve to estimate 50% inhibitory concentration (IC50). |
| Affinity data for this assay | |
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