| Assay Method Information | |
| | In Vitro assay |
| Description: | The determination of IC50 of the inhibiting effect to kinase ALK 96-well plates was coated under 37° C. with coating buffer (125 ul/well) overnight, and coating buffer was polypeptide substrate [Poly (4:1 Glu, Tyr) Peptide, available from SignalChem] containing 2.5 ug/well ALK kinase in PBS. Then, each well was washed with 200 ul of buffer (PBS containing 0.05% Tween 80), and placed at 37° C. for at least 2 hours to dryness. Serial diluted test compounds in different concentrations (compounds prepared in any one of Examples 1 to 16, dissolved in DMSO) were added to each well in 5/well, followed by addition of kinase buffer (25 mM Hepes, pH 7.5, 5 mM MnCl2, 5 mM MgCl2), 0.3 mM ATP, and 100 ng/well recombinant human ALK (Abnova Corporation), to a total volume of 100 ul each well. After kept at 30° C. for 15 minutes, the reaction mixture was removed, and washed with 200 ul of wash buffer (PBS containing 0.05% Tween 80) for 5 times. |
| Affinity data for this assay | |
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