| Assay Method Information | |
| | Inhibition of SIK; Abl and Src Kinases |
| Description: | Briefly, a radiometric protein kinase assay (33PanQinase® Activity Assay) was used for measuring the kinase activity of the five protein kinases. All kinase assays were performed in 96-well FlashPlates™ from PerkinElmer (Boston, MA, USA) in a 50 uL reaction volume. The reaction cocktail was pipetted in four steps in the following order:25 uL of assay buffer (standard buffer/[gamma-33P]-ATP)10 uL of ATP solution (in water)5 uL of test compound (in 10% DMSO)20 uL enzyme/substrate mixThe assay for all protein kinases contained 70 mM HEPES-NaOH pH7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, ATP (variable concentrations, corresponding to the apparent ATP-Km of the respective kinase, see Table 2A), [gamma-33P]-ATP (approx. 8×105 cpm per well), protein kinase (variable amount, see Table 2A), and substrate (variable amounts, see Table 2A). |
| Affinity data for this assay | |
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