| Assay Method Information | |
| | In Vitro Tyrosine Kinase Enzyme Assay |
| Description: | Protein kinase assays were performed at ambient temperature for 20-40 min (depending on the target) in a final volume of 25 μl according to the following assay reaction recipe: 5 ml of diluted active protein kinase target (~10-50 nM final protein concentration in the assay), 2.5 μl of peptide substrate, 3. μl of kinase assay buffer, 5 μl of compound or 10% DMSO, and 5 μl of 33P-ATP (250 μM stock solution, 0.8 mCi). The assay was initiated by the addition of 33P-ATP and the reaction mixture was incubated at ambient temperature for 20-40 min, depending on the protein kinase target. After the incubation period, the assay was terminated by spotting 10 μl of the reaction mixture onto Multiscreen phosphocellulose P81 plate. The Multiscreen phosphocellulose P81 plate was washed three times for approximately 15 min each in a 1% phosphoric acid solution. The radioactivity on the P81 plate (EMD Millipore Corporation, Billerica, MA) was counted in the presence of scintillation fluid in a Trilux scintillation counter (GMI Inc., Ramsey, MN). |
| Affinity data for this assay | |
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