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| | KinEASE Assay |
| Description: | IC50 determinations for activated Akt1 were measured with the KinEASE assay (Cisbio) according to the manufacturer’s instructions. The kinases Akt1 (batch no. D8MN034U-L) was purchased from Millipore. 5 μL kinase solution (0.12 nM for Akt1) and 2.5 μL inhibitor solution (8% DMSO in HTRF buffer) were pre-incubated for 1 h before the reaction was started by addition of 2.5 μL of a solution containing ATP and substrate peptide. The final ATP and substrate concentrations were set at their respective Km values (50 μM and 250 nM, respectively, for Akt1). The biotinylated STK3 substrate peptide is then phosphorylated by Akt1 for 45 min. After completion of the reaction, 10 μL of a solution with an anti-phosphoserine/-threonine antibody labeled with Europium Cryptate and Streptavidin labeled with the fluorophore XL665 (0.5 μM for Akt1) were added simultaneously. The FRET between Europium Cryptate and XL665 was measured to quantify the extent of substrate pep |
| Affinity data for this assay | |
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