| Assay Method Information | |
| | Kinase Assay |
| Description: | The kinase assay is performed as 384-well flashplate assay.1 nM IKKε, 800 nM biotinylated IκBα(19-42) peptide (biotin-C6-C6-GLKKERLLDDRHDSGLDSMKDEE) and 10 μM ATP (with 0.3 μCi of 33P-ATP/well) are incubated in a total volume of 50 μl (10 mM MOPS, 10 mM magnesium acetate, 0.1 mM EGTA, 1 mM dithiothreitol, 0.02% of Brij35, 0.1% of BSA, 0.1% of BioStab, pH 7.5) with or without test substance at 30° C. for 120 min. The reaction is stopped using 25 μl of 200 mM EDTA solution, filtered off with suction after 30 min at room temperature, and the wells are washed 3 times with 100 μl of 0.9% NaCl solution. The non-specific proportion of the kinase reaction (blank) is determined using 3 μM EMD 1126352 (BX-795). Radioactivity is measured in the Topcount. IC50 values are calculated using RS1. |
| Affinity data for this assay | |
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