| Assay Method Information | |
| | Inhibition of SIK; Abl and Src Kinases by Other Kinase Inhibitor |
| Description: | The IC50 for the inhibition of ABL1 by dasatinib, by C7 and by B3 is approximately 1.5 nM, 5.1 nM and 1.6 nM, and of SRC is 1.5 nM, 1.5 nM and 1.5 nM; each, respectively for dasatinib, C7 and by B3 (Table 3A). The compound C12 is also a strong inhibitor of SRC (<100 nM IC50), and is selective to SRC over ABL1. 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 mix. The 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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