CHEMBL3887652

ABL1 Kinase Mobility-Shift Assay: Axitinib was tested with the Caliper LabChip3000 assay (Caliper Life Science, Hopkinton, Mass.), which is a mobility-shift assay (MSA) that combines the basic principles of capillary electrophoresis in a micro-fluidic environment. Axitinib was prepared in 100% DMSO, diluted to 25% DMSO with 20 mM HEPES pH 7.5, and added to the reaction for a final DMSO concentration of 6%. Inhibitor concentrations varied from 1.0 uM to 0.00003 uM. Typical reactions were 20 uL, contained ABL1 or ABL1 [T315I], ATP (ABL1; 25 uM and ABL1 [T315I]; 5 uM), 1.0 uM ABLtide, 5 mM MgCl2, 2 mM DTT, 0.01% Triton X-100 (Sigma-Aldrich, St. Louis, Mo.), 6% DMSO in 20 mM HEPES pH 7.5. The mixture was incubated in a 384-well polypropylene plate at room temperature for an hour and terminated by the addition of 60 uL of QuickScout Screening Assist MSA Buffer (Carna Biosciences, Kobe, Japan).