| Assay Method Information | |
| | Kinase Inhibition Assay |
| Description: | The assay used was the HotSpot assay (Reaction Biology Corp).Compounds were tested in 10-dose IC50 mode with a 3-fold serial dilution starting at 10 μM. Control Compound, Staurosporine, was tested in 10-dose IC50 mode with 4-fold serial dilution starting at 20 μM. Reactions were carried out at 10 μM ATP.ReagentsBase Reaction buffer; 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO.*Required cofactors are added individually to each kinase reactionReaction Procedure1. The peptide substrate was freshly prepared in Base Reaction Buffer2. Any required cofactors were delivered to the substrate solution above3. The human recombinant Casein kinase 16 was delivered into the substrate solution and gently mixed4. The compounds in DMSO were delivered into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), and incubated for 20 minutes at room temperature5. 33P-ATP (specific activity 10 mCi/mL) together with ATP (10 μM) was delivered into the reaction mixture6. The kinase reaction was incubated for 2 hours at room temperature7. The reaction mixtures were spotted onto P81 ion exchange paper8. The kinase activity was detected by measuring 33P-ATP-labelled product peptide using a filter-binding method. |
| Affinity data for this assay | |
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