| Assay Method Information | |
| | Enzyme Assay |
| Description: | In vitro IDO1 enzymatic activity was determined in a mixture of 50 mM MES buffer at pH 6.5; 200 nM human IDO enzyme, 150 μM L-Tryptophan, 2250 units/mL Catalase, 20 mM ascorbic Acid and 10 μM Methylene Blue. The compounds were initially prepared in DMSO at 10 mM, then diluted in MES buffer to desired concentration. 25 μL compounds were added to 96 well plate, followed by addition of 25 μL 33.68 ng/μL IDO1 in each well. The mixture was centrifuged for 1 minute, then pre-incubated at room temperature for 30 minutes. The reaction was started by the addition of 50 μL mixture of 300 μM L-Tryptophan, 4500 units/mL catalase and 20 μM methylene blue in 50 mM pH6.5 MES buffer, and 40 mM ascorbic Acid in 0.405M pH 8.0 Tris HCl buffer. The resulting reaction mixture was incubated at 25° C. for 40 minutes. The reaction was terminated by adding 50 ul of 30% (w/v) trichloroacetic acid. The sample was further incubated for 30 min at 60° C. and centrifuged at 2000 rpm for 5 min to remove precipitated protein. The supernatant was used to mix with an equal volume of Ehrlich's reagent (2% w/v p-dimethylaminobenzaldehyde in glacial acetic acid), then mixture was incubated at room temperature for 10 minutes. OD value was read at 490 nm in a spectrophotometer. |
| Affinity data for this assay | |
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