| Assay Method Information | |
| | In Vitro AMPK Activation Assay |
| Description: | The in vitro AMPK activation assay is performed in a volume of 30 ul in a 384-well plate. Enzyme reactions were assembled in the microtiter plate by adding 15 ul of 2X enzyme in assay buffer (20 mM HEPES, pH 7.3, 5 mM MgCl2, 3 mM DTT, 0.01% Brij 35 and CamK Kinase, to activate AMPK) to wells which contained either DMSO or compound. The reaction was initiated with the addition of 15 ul 2x substrate mixture containing 200 uM ATP, and 3.0 uM fluorescently labeled SAMS (5-FAM-HMRSAMSGLHLVKRR-COOH) in assay buffer. After 45-minute incubation at 25° C., the reaction was stopped by the addition of 70 ul stop buffer (100 mM HEPES, pH 7.3, 40 mM EDTA, 0.015% Brij 35). Phosphorylated 5-FAM SAMS product is assessed using a Caliper EZ Reader LabChip microfluidics reader. Product conversion is determined by calculating the peak heights of the substrate and product and reporting the product/(product+substrate) peak ratio. The 10-point titration data were expressed as % maximum AMP activation. The results were plotted using 4 parameter fit and the inflection point reflecting 50% of the maximum activation was reported as the EC50. |
| Affinity data for this assay | |
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