| Assay Method Information | |
| | Time-Resolved Fluorescence-based Lanthascreen Assay |
| Description: | Inhibitions studies were performed using a time-resolved fluorescence-based Lanthascreen assay. Phosphorylation of a fluorescein-labelled substrate peptide is measured using terbium-labeled phosphospecific antibodies. Terbium is excited at 340 nm and the FRET energy transfer to fluorescein is measured at 495 and 520 nm. The emission ratio between 520 and 495 is a measure of the level of phosphorylation of the substrate by the kinase.Kinase inhibition assays (10 L) were performed at 20 ° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 M) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkB ± substrate (DRHDSGLDSMKDE), 20 M ATP, 2 nM or 8 nM IKK µ or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35. |
| Affinity data for this assay | |
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