| Assay Method Information | |
| | In Vitro AAK1 Enzyme Activity Assay |
| Description: | Compound stock solution (concentration: 10 mM, dissolved in DMSO) was diluted with DMSO to 0.2 mM and then diluted with DMSO in 5-fold gradient to obtain compound solutions with 10 concentrations. Subsequently, the compound solutions with different concentrations were diluted 50-fold in 1×kinase reaction buffer (containing 40 mM Tris, 20 mM MgCl2, 0.1% BSA and 0.5 mM DTT) for later use. AAK1 (Signalchem, Cat #A01-11G-10) was diluted with 1×kinase reaction buffer to 2-fold the final concentration (final concentrations: 30 nM and 28 nM). AAK1 was added to a 384-well white plate at 2 μL/well, and the compounds were then added at 1 μL/well. The plate was sealed with a plate-sealing film, centrifuged at 1000 rpm for 30 seconds and then placed at room temperature for 10 minutes. A mixed solution of ATP (Promega, Cat #V914B) and substrate Micro2 (GenScript, Cat #PE0890) was formulated at 4-fold the final concentration (for AAK1, the corresponding final concentrations of ATP: 15 μM and 5 μM, and the corresponding final concentration of Micro2: 0.1 mg/mL). To the reaction plate was added the mixed solution of ATP and the substrate at 1 μL/well. |
| Affinity data for this assay | |
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