| Assay Method Information | |
| | Inhibitory Activity Against PKMYT1 Kinase |
| Description: | The reaction buffer was used as a composition of 200 mM Tris-HCl pH 7.4, 100 mM MgCl2, 0.5 mg/mL BSA, 0.25 mM DTT, and the reactions of all the tests were carried out on the reaction buffer. The compound was diluted in 12 steps using a serial dilution method from a 10 mM DMSO stock, and the enzyme activity was measured at a final compound concentration of 10˜0.00005645 μM. During the test, the enzyme activity was confirmed using in vitro ADP-Glo™kinase assay (Promega) after reacting with the proper concentration of PKMYT1 enzyme, purified ATP, and enzyme substrate (CDK1) at 25° C. for 1 hour. At a ratio of 2:2:1, an enzyme-active reaction solution, an ADP-Glo reaction solution, and an enzyme-active detection solution were reacted to measure the inhibition of the enzyme's activity with luminescence. Based on the fluorescence of the enzyme activity of the solvent control that was not treated with the compound, the degree of enzyme activity inhibition was calculated according to the treatment concentration of each compound, and the concentration of each compound that inhibits 50% of enzyme activity was determined as IC50 (nM) value and the value was obtained by using GraphPad Prism 8.3.0 (GraphPad software Inc., San Diego) software. |
| Affinity data for this assay | |
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