| Assay Method Information | |
| | The determination of IC50 of the inhibiting effect to kinase ALK |
| Description: | 96-well plates was coated under 37° C. with coating buffer (125 μl/well) overnight, and coating buffer was polypeptide substrate [Poly (4:1 Glu, Tyr) Peptide, available from SignalChem] containing 2.5 μg/well ALK kinase in PBS. Then, each well was washed with 200 μl 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 μl/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 μl each well. After kept at 30° C. for 15 minutes, the reaction mixture was removed, and washed with 200 μl of wash buffer (PBS containing 0.05% Tween 80) for 5 times.100 μl/well of mouse anti-phosphotyrosine monoclonal antibody (clone 4G10, purchased from EMD Millipore Corporation) were used in the detection of phosphorylated peptide substrate. Monoclonal antibody was diluted at 1:500 with PBS containing 4% bovine serum albumin. After incubated at room temperature for 30 minutes, the antibody solution was removed, and each well was washed with 200 μl of wash buffer (PBS containing 0.05% Tween 80) for 5 times.100 μl/well of secondary antibody (anti-mouse IgG) was added. The secondary antibody was diluted at 1:1000 with PBS containing 4% bovine serum albumin, incubated for 30 minutes at room temperature; and the wells were washed again as said above.100 μl/well of TMB substrate solution was used for color development, and the same volume of 0.18 M H2SO4 was added to terminate the color development. Finally, the absorbance at 450 nm or 490 nm was read, and the IC50 was calculated. |
| Affinity data for this assay | |
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