Assay Report Card

ID: ALA3705728

Type: Binding

Description: Kinase Assay: Inhibition of C-kit kinase activity by compounds disclosed herein was quantified by measuring the amount of [33P] incorporated into the substrate in the presence of a test compound. Briefly, a reaction mixture of 25 uL final volume containing 5 ng of C-kit kinase (obtained by purifying recombinant N-terminal 6xHis-tagged C-kit kinase domain construct expressed from baculovirus), 5 ug of the substrate [Poly (Glu-Tyr, 4:10, Sigma], kinase reaction buffer (8 mM MOPS/NaOH pH7.0, 0.2 mM EDTA, 10 mM MnCl2, 0.5% glycerol, 0.001% Brij-35, 0.01%, 2-mercaptoethanol, 0.1 mg/ml BSA, 100 uM ATP, 0.1 uCi per well [33P]-gamma-ATP (2,500-3,000 Ci/mmol)), and a test compound (diluted from 9 uM to the desired final concentration by 4% DMSO) or DMSO (as the control) was incubated at 30C. for 30 minutes. The reaction was stopped by adding 5 uL of 3% phosphoric acid solution. The resultant solution (30 uL) was later harvested by a UniFilter Plate GF/B (PerkinElmer).

Organism: Homo sapiens