| Assay Method Information | |
| | GPa Inhibition Assay |
| Description: | RMGPa (Rabbit Muscle Glycogen Phosphorylase a) activity was measured in the direction of glycogen synthesis by the formation of inorganic phosphate from glucose-1-phosphate using a 384 well plate at 22 deg with a 30 min incubation time. Phosphate was measured at 620 nm, 5 min after the addition of ammonium molybdate and malachite green. Test compounds were added to the assay. Compounds were tested against a caffeine standard in 11 point concentration-response curve in duplicate on two separate occasions. Data was analyzed using GraphPad Prism v.4.03.A nonlinear regression (curve fit) analysis with a sigmoidal dose-response equation (variable slope) was applied to generate IC50 and Hill slope values. The reported IC50 had a Hill slope between 0.7 and 1.2 and a Z value of ~0.8. Compounds were screened with maximal concentrations of 222 uM. The assay was carefully monitored for signs of compound insolubility. The results are presented as mean values from 4 determinations. Samples used in screening were of 98-100% purity. |
| Affinity data for this assay | |
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