| Assay Method Information | |
| | Kinase Inhibition Assay |
| Description: | Adapta Kinase Assay Method: Adapta universal kinase assay method is a uniform fluorescence-based immunoassay for detecting ADP. In contrast to ATP consumption assay, Adapta assay method was very sensitive to ADP formation, with most signal changes occurring when initially 10-20% ATP was converted to ADP. This made Adapta universal kinase assay method very suitable for low-activity kinases. All assays were performed at room temperature (about 21 C.) and were linearly related to time and enzyme concentration under the conditions used. 1 kinase reaction buffer was prepared from 5 kinase buffer S stock solution (listed above), and 10 mL of 1 kinase reaction buffer was prepared by the addition of 2 mL of 5 stock solution to 8 mL of H2O. To the 1 kinase reaction buffer was added 20 μL of 1 M DTT. A kinase reaction was performed in a low-volume 384-well plate in a volume of 10 μL. Typically, a Greiner plate was used (Catalog #3674 #, low volume, white wall, 784075). Other untreated assay plates that were not tested could also be suitable. The substrate concentration in the assay was 100 μM, and the 1 kinase reaction buffer consisted of 50 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35 and 2 mM DTT, as well as any other additives that could be necessary for the particular kinase. The kinase reaction was allowed to proceed for 1 h at room temperature, followed by the addition of a formulation of kinase quenching buffer (EDTA; 30 mM), Eu-labeled antibody (6 nM), and tracer (18.9 nM) in TR-FRET dilution buffer (5 μL). The final concentration of the antibody in the assay wells was 2 nM, the final concentration of the tracer was 6.3 nM, and the final concentration of EDTA was 10 mM. The plate was allowed to equilibrate at room temperature for at least 30 min and then read on a plate reader configured for Adapta TR-FRET. The data provided in this document were generated by using BMG LABTECH PHERAstar plate reader and using appropriate filters and instrument settings for Adapta . |
| Affinity data for this assay | |
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