| Assay Method Information | |
| | in vitro AMPK activation assays |
| Description: | The test compounds were dissolved in 100% DMSO at a concentration of 10 mM and in a first step diluted in DMSO to a concentration of 5 mM, followed by serial dilution steps in 100% DMSO. Dilution factor and number of dilution steps may vary. Typically 8 different concentrations by 1:5 dilutions were prepared, further dilutions of the substances were carried out with test buffer (20 mM Hepes (pH 7.0), 15 mM MgCl2, 0.025% BSA, 0.01% Brij 35) until a concentration was reached which was 5 times above the final test concentration. 2 μl aliquots of these dilutions were transferred into a 384-well Optiplate (Perkin Elmer, #6007290). Typically the start concentration for serial dilutions in the assay is 10 μM. Typically AMPK was diluted to 25 μg/ml in the test buffer and 4 μl of this dilution were used in the kinase test (final concentration of AMPK is 10 μg/ml in a total volume of 10 μl for the kinase reaction). Kinase concentrations may vary depending on activity of the preparation batches. After 10 minutes incubation at room temperature 4 μl of a mix containing 2.5 μM substrate (H-His-Met-Arg-Ser-Ala-Met-Ser-Gly-Leu-His-Leu-Val-Lys-Arg-Arg-OH Trifluoroacetate salt/HMRSAMSGLHLVKRR from Bachem, Cat. No. H5938) and 75 μM ATP in test buffer were added to each well and the incubation was continued for 60 minutes at room temperature. Positive controls are the reaction mixtures that contain no test substance; negative controls (blanks) are reaction mixtures that contain no AMPK enzyme. After 60 minutes, 10 μl ADP-Glo solution (ADP-Glo Reagent #V912B Promega) (heated to room temperature) were added to each well and incubation was continued for 40 minutes. Then 20 μl Kinase detection mix (Detection Buffer #V913B Promega; Kinase Detection Substrate #V914B Promega) were added and incubated for additional 40 minutes at room temperature. All incubations were done in sealed plates in the dark. The plates were read with an Envision Luminescence Reader (Perkin-Elmer). |
| Affinity data for this assay | |
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