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| | KinEASE-STK Assay |
| Description: | IC50 determinations for TBK1 were performed with the KinEASE-STK assay from Cisbio according to the manufacturer's instructions. A biotinylated substrate peptide (STK3 from Cisbio) was phosphorylated by TBK1. After completion of the reaction, an antiphosphoserine/-threonine antibody labeled with europium cryptate and streptavidin labeled with the fluorophore XL665 were added. FRET between europium cryptate and XL665 was measured to quantify the phosphorylation of thesubstrate peptide. ATP concentrations were adjusted to 30 μM while a substrate concentration of 1 μM was used. Kinase and inhibitor were preincubated for 30 min before the reaction was started by addition of ATP and substrate peptide. A Tecan infinite M1000 plate reader was used to measure the fluorescence of the samples at 620 nm (Eulabeled antibody) and 665 nm (XL665 labeled streptavidin) 60 μs afterexcitation at 317 nm. |
| Affinity data for this assay | |
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