| Assay Method Information | |
| | In Vitro Kinase Assay |
| Description: | Inhibition of kinase activity was measured in a DELFIA assay (Perkin-Elmer). The substrate poly (Glu4, Tyr) was immobilized onto black high-binding polystyrene 96-well plates (Nunc Maxisorp). The recombinant enzymes were preincubated with inhibitor and Mg-ATP on ice in polypropylene 96-well plates for 4 min, and then transferred to the substrate coated plates. After the kinase reaction, phosphorylated product was detected by incubation with Europium-labeled anti-phosphotyrosine MoAb. The bound MoAb was detected by time-resolved fluorescence in a Gemini SpectraMax reader (Molecular Devices). Inhibitors were tested at seven different concentrations each in triplicate. IC50s were calculated in a four parameters equation curve plotting inhibition (%). |
| Affinity data for this assay | |
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