| Assay Method Information | |
| | Enzyme Assay |
| Description: | In vitro TDO enzymatic activity was determined in a mixture of 50 mM potassium phosphate buffer, pH 6.5; 200 nM human TDO enzyme, 300 μM L-Tryptophan, 0.2 mg/mL Catalase, 20 mM ascorbic Acid and 20 μM Methylene Blue. 100× compounds were prepared in DMSO from 1 mM, then diluted three fold, 8 doses in total. 2 μL compounds were added to 96 well plate, followed by addition of 100 μL 400 nM TDO and 0.4 mg/ml catalase in each well. The mixture was centrifuged for 1 minute, then pre-incubated at room temperature for 10 minutes. The reaction was started by the addition of 100 μL mixture of 600 μM L-Tryptophan, 40 μM methylene blue and 40 mM ascorbic acid in 50 mM potassium phosphate buffer, pH 6.5. The resulting reaction mixture was shaken 30 secs and Kineticly read the plate in SpectraMax 384 at OD321 nm for 20 mins at RT. Copy slope data from Synergy program, and convert slope values to inhibition values. Percent inhibition=(max−conversion)/(max−min)*100. max stands for high control; min stands for low control. Fit the data in GraphPad Prism5.0 to obtain IC50 values. |
| Affinity data for this assay | |
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