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Assay Method Information

Assay Name:  ABL1 Kinase Mobility-Shift Assay
Description:  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).
Affinity data for this assay
 

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Last update November 1, 2007
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