| Assay Method Information | |
| | TDO2 Enzymatic Assay |
| Description: | The TDO2 enzymatic assay was performed by UV absorption using a recombinant TDO2 and L-Tryptophan substrate. The UV absorption signal at 321 nm is correlated with the amount of N-formylkynurenine reaction product of TDO2. The compounds were diluted in 10% DMSO and 5 μL of the dilution was added to a 100 μL reaction so that the final concentration of DMSO is 0.5% in all of reactions. All of the reactions were conducted at room temperature. The 100 μL reaction mixture in TDO2 Assay Buffer contains 50 nM TDO2, the indicated amount of the inhibitor, 200 μM tryptophan, and the coupled reaction components. The reaction mixture incubated for 75 min prior to reading the UV absorption signal. For the negative control (blank), 5 μl of the assay buffer was added instead of the TDO2.Absorption signals were measured using a Tecan Infinite M1000 plate reader. Binding experiments were performed in duplicate at each concentration. The percent activity in the presence of each compound was calculated according to the following equation: % activity=[(A−Ab)/(At−Ab)]×100, where A=the absorption signal in the presence of the compound, Ab=the absorption signal of blank, At=the absorption signal in the absence of the compound. The percent inhibition was calculated according to the following equation: % inhibition=100−% activity. The IC50 was calculated using non-linear regression in Graph Pad Prism 4. |
| Affinity data for this assay | |
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