| Assay Method Information | |
| | LCK Biochemical Assay |
| Description: | The standard protocol below describes the standard HotSpot Kinase Assay run by the Reaction Biology Corporation as a service.The substrate (poly(EY) (Glu:Tyr (4:1)) and the cofactors were prepared in fresh Reaction Buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 2% DMSO). To the substrate solution was then added the enzyme (LCK; recombinant human full-length (Genbank Accession #NP_005347); C-terminal His-tagged, expressed in insect cells), and the resulting solution was then gently mixed. The test compounds were dissolved in DMSO and then delivered into the kinase reaction mixture using acoustic liquid handling technology (Echo550; nanoliter range). The reaction mixture was incubated for 20 min at RT and then treated with 33P-ATP to initiate the reaction. After 2 hours of incubation at RT, kinase activity was detected using Reaction Biology Corporation's proprietary P81 filter-binding method. |
| Affinity data for this assay | |
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