| Assay Method Information | |
| | AKT Kinase Activity Assay |
| Description: | a) first, a compound stock solution (10 mM DMSO solution) was diluted with DMSO to obtain a 100 µM compound solution, the compound solution was diluted with the 1× kinase reaction buffer to obtain a 2.5 µM compound working solution (containing 2.5% DMSO). A 2.5% DMSO solution was prepared from the 1× kinase reaction buffer, and the 2.5 µM compound working solution was diluted 7 times with the 2.5% DMSO solution according to a 4-fold gradient to obtain compound working solutions at 8 concentrations (2500 nM, 625 nM, 156 nM, 39 nM, 9.8 nM, 2.4 nM, 0.6 nM, and 0.15 nM). Except for control wells, 4 µL of diluted compound working solution was placed in each reaction well, and 4 µL of previously prepared 2.5% DMSO/kinase buffer was placed in each control well. b) 2 µL of previously prepared TK-biotin substrate solution (the concentration of the substrate for enzyme screening is shown in Table 1) was placed in each reaction well. c) 2 µL of previously prepared enzyme solution (the concentration of the enzyme is shown in Table 1) was placed in each reaction well except for negative wells, and 2 µL of 1× kinase reaction buffer corresponding to the enzyme was placed in each negative well to make up the volume. The plate was sealed with a sealing film, and the reaction solution was mixed until uniform and incubated at the room temperature for 10 min to allow the compound to fully react with and bind to the enzyme. d) 2 µL of ATP solution was placed in each reaction well to initiate a kinase reaction (the concentration of ATP for enzyme screening and reaction time are shown in Table 1). e) 5 min before the kinase reaction was completed, an assay solution was prepared. Streptavidin-XL665 and a europium-labeled tyrosine kinase substrate antibody (1: 100) assay solution (the concentration of the assay reagent is shown in Table 1) were prepared from the assay buffer in the kit. f) After the kinase reaction was completed, 5 µL of diluted streptavidin-XL665 was placed in each reaction well and mixed with the reaction solution until uniform, and the diluted europium-labeled tyrosine kinase substrate antibody assay solution was immediately added.g) The plate was sealed, the reaction solution was mixed until uniform and reacted at the room temperature for 1 h, and fluorescence signals were detected by using an ENVISION (Perkinelmer) instrument (320 nm stimulation, 665 nm, 615 nm emission). |
| Affinity data for this assay | |
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