| Assay Method Information | |
| | HotSpot Kinase Assay |
| Description: | PKMYT1: Inhibition of PKMYT1 kinase activity by test compounds was measured by the HotSpot Kinase Assay at Reaction Biology Corporation (Malvern, Pa.). Briefly, Myelin Basic Protein substrate was prepared in Reaction Buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO). PKMYT1 kinase was delivered into the substrate solution and gently mixed. Test compounds in 100% DMSO were added into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range) and incubated for 20 min at room temperature. 33P-ATP was delivered into the reaction mixture to initiate the reaction. Reactions were carried out at 10 μM ATP. After a 2 hour incubation at room temperature, kinase activity was detected by P81 filter-binding method. Compounds were tested in 10-dose IC50 mode with a 3-fold serial dilution. |
| Affinity data for this assay | |
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