Figure 4. Characterization of Abl kinase using the BacKin assay.

Mean fluorescence was converted into fraction of phosphorylation, assuming maximum response as 100% of abltide phosphorylation. Each data point is the average of three independent experiments±SD (A) Abltide phosphorylation catalyzed by increasing concentrations of Abl kinase in the presence of 500 µM ATP and incubated for 30 min at 37°C. The kinase concentration required to achieve half of the maximum response, K_D±SD, was calculated by fitting a nonlinear sigmoidal curve. (B) ATP concentration effect on abltide phosphorylation catalyzed by 0.5 U/mL Abl Kinase for 30 min at 37°C. The ATP concentration required to achieve half of the maximum response, K_D±SD, was calculated by fitting a nonlinear sigmoidal curve. (C) Time-course of abltide phosphorylation catalyzed by 0.5 U/mL Abl kinase in the presence of 500 µM ATP at 37°C obtained with BacKin assay (circles) or with LC-MS assay (squares). The time required to phosphorylate half of the abltide molecules, t_1/2±SD, was calculated by fitting a pseudo-first order association kinetics.