| Assay Method Information | |
| | In Vitro Inhibitory Activity Evaluation of Human Wild-type and V804M Mutant RET Kinase |
| Description: | The inhibitory activity of the test compounds against human wild-type and V804M mutant RET kinase was evaluated by measuring IC50 values in a 33P-labeled kinase activity assay (Reaction Biology Corp). Buffer conditions: 20 mM hydroxyethyl-piperazine-ethanesulfonic acid (Hepes) (pH 7.5), 10 mM MgCl2, 1 mM ethylene glycol-bis(aminoethyl ether)-tetraacetic acid (EGTA), 0.02% polyoxyethylene lauryl ether (Brij35), 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM dithiothreitol (DTT), and 1% DMSO. Compound handling: testing compounds were dissolved in 100% DMSO to specific concentration. Serial dilution can be conducted using Integra Viaflo Assist in DMSO. Procedures: the substrate was dissolved in freshly prepared Reaction Buffer, the test kinase was added into the substrate solution and gently mixed. Compounds in DMSO were added into the above kinase reaction mixture by Acoustic technology (Echo550) and incubate for 20 minutes at room temperature. The concentrations of the compounds in the reaction solutions were 3 μM, 1 μM, 0.333 μM, 0.111 μM, 0.0370 μM, 0.0123 μM, 4.12 nM, 1.37 nM, 0.457 nM and 0.152 nM. After incubation for 15 min, 33P-ATP (Specific activity of 0.01 μCi/μL, Km concentration) was added into the reaction mixture to initiate the reaction. The reaction was conducted for 120 minutes at room temperature, and radioactivity was detected by filter-binding method. Kinase activity data can be expressed as the percent remaining kinase activity in test samples compared to vehicle (DMSO only) reactions. IC50 values and curve fits can be obtained using Prism4 (GraphPad Software). |
| Affinity data for this assay | |
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