| Assay Method Information | |
| | ALK HTRF Assays |
| Description: | The ALK kinase activity of five target compounds and three positive compounds were evaluated using standard homogeneous time-resolved fluorescence (HTRF) assay (High Fluorescence Resonance Energy Transfer). The test compounds or reference controls were dissolved in DMSO as stock solution. The DMSO solution was serial diluted (duplicated with 3-fold dilution) to make that the concentrations were from 10 然 to 0.17 nM in 11 doses. The enzyme reaction mixture of ALK WT/ALK C1156Y/ALK L1196M for HTRF assay contained 0.5 nM ALK WT/0.15 nM ALK C1156Y/0.15 nM ALK L1196M, 1 然 biotin-TK peptide, and 30 然 ATP in a kinase reaction buffer containing 50 mM HEPES (pH 7.5), 5 mM MgCl2, 0.01% BSA, 0.1 mM Orthovanadate at a final volume of 10 無. The enzyme reaction were carried out at room temperature in white Proxiplate 384-Plus plate (PerkinElmer) for 90 min. 10 無 of the detection reagents contained 6.7 nM TK-Antibody and 16.67 nM XL665 in a detection buffer containing 50 mM HEPES (pH 7.5), 0.8 |
| Affinity data for this assay | |
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