| Assay Method Information | |
| | Biochemical Assay |
| Description: | The reaction procedure was conducted as follows:1) Prepare substrate in freshly prepared Reaction Buffer.2) MnCl2 (2 mM) was added as a co-factor listed as above.3) Deliver kinase into the substrate solution and gently mix.4) Deliver compounds in 100% DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 min at RT.5) Deliver 33P-ATP (specific activity 10 μCi/μL) into the reaction mixture to initiate the reaction.6) Incubate for 2 hours at RT.7) Detect radioactivity by the filter-binding method.8) Kinase activity data was expressed as the percent remaining kinase activity in the test sample compared to vehicle (DMSO) reactions. Curve fits were performed where the enzyme activities at the highest concentration of compounds were less than 65%. IC50 values and curve fits were obtained using Prism (GraphPad Software). |
| Affinity data for this assay | |
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