| Assay Method Information | |
| | Radiometric Protein Kinase Assay |
| Description: | A radiometric protein kinase assay was used for measuring the kinase activity of the Dog (Canis lupus familiaris) IRAK4, JAK1, JAK2, SYK kinases. All kinase assays were performed in 96-well FlashPlates in a 50 μl reaction volume. The reaction cocktail was pipetted in four steps in the following order:20 μl of assay buffer (standard buffer)5 ml of test compound (in 10% DMSO)20 μl enzyme/substrate mixμl of ATP solution (in H2O) The reaction cocktails were incubated at 30° C. for 60 minutes. The reaction was stopped with 50 μl of 2% (v/v) H3PO4, plates were aspirated and washed two times with 200 μl 0.9% (w/v) NaCl. Incorporation of 33Pi was determined with a microplate scintillation counter.IC50 calculation: the residual activity (in %) for each well of a particular plate was calculated by using the following formula: Res. Activity (%)=100×[(cpm of compound−low control)/(high control−low control)] |
| Affinity data for this assay | |
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