| Assay Method Information | |
| | C-abl Kinase Assay |
| Description: | c-Abl kinase activity was measured by Promega s ADP-Glo Assay. In this assay, His-tagged recombinant human ABL1 (0.25 ng/ l) is incubated with 5 L of compounds (0.5% DMSO), 5 L of Abltide (0.01 g/ l) and 5 L of ATP (25 M) in buffer (50 mM HEPES,7.5; 10 mM MgCl2; 1 mM EGTA; 0.05% BSA; 0.01% Tween-20; 2 mM DTT.). The assay was started by incubating the reaction mixture in a 96-well plate at 30 C. for 30-min. After the incubation, 25 L ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 L per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and Sigma Plot 13.0. |
| Affinity data for this assay | |
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