| Assay Method Information | |
| | AKT1 E17K Kinase Biological Activity Assay |
| Description: | Purified human recombinant kinase full-length AKT1 E17K was expressed in insect cells and activated in vitro by PDPK1. UlightTM-CREBtide (PerkinElmer, catalog number TRF0107) was used as the peptide substrate. The reaction buffer consisted of 50 mM HEPES pH 7.5, 10 mM MgCl2, 0.01% Triton X-100, 0.01% BSA, 2 mM DTT, 0.6 nM AKT1 E17K, 50 nM UlightTM-CREBtide and either 50 μM ATP or 2 mM ATP. The compound solution was prepared as 10 mM DMSO stock. For the assay, a 1:3 serial dilution 10-pt dose response of test compounds, in duplicate, for each concentration were dispensed into a 384-well plate (Corning, catalog number 4513). Final DMSO concentration in the assay was 1%. The enzyme mixture (5 μL, 1.2 nM) were added and the mixture was incubated for 15 min at RT prior to the start of the kinase reaction by the addition of 5 μL substrate mixture (100 nM UlightTM-CREBtide). The reaction was stopped after 60 min of incubation at RT by the addition of 10 μL of Detection Mix (PerkinElmer, #CR97-100) consisting of Europium-anti-phospho-CREB (Ser133) (Perkin Elmer, catalog numberTRF0200). Incubation with the detection mix for a further 60 min allowed binding of antibody to the phospho-UlightTMCREBtide. The fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured using Envision reader. The ratio of the emissions at 665 nm and at 620 nm provided the measure for the amount of phosphorylated CREBtide product of AKT1 E17K kinase activity. To determine the IC50 values, the data was normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components but no enzyme = 100% inhibition) and IC50 values were calculated by a 4-parameter fit using an in-house developed protocol. |
| Affinity data for this assay | |
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