Biochemical Assay Kit for Human TDO (hTDO)
NFK Green™ Assay Technology
Tryptophan 2,3-dioxygenase (TDO) is an enzyme that converts L-tryptophan into NFK, which is the first step in the kynurenine pathway of tryptophan degradation. TDO is an important drug target for cancer immunotherapy and neurodegenerative diseases. NFK can form a complex with the chemical probe NFK Green™ and can thereby be detected in a fluorescence assay. The human TDO (hTDO) biochemical assay kit can be used to determine the potency of TDO inhibitors.
Schematic representation of the NFK Green™ assay principle. After enzymatic conversion of L-tryptophan into NFK, the reaction is stopped by addition of NFK Green™, which yields a fluorescent complex that can be detected at the indicated wavelengths.
Dose-response curve for the reference TDO inhibitor in the NFK Green™ hTDO assay.
Kits contents
| Component | Description | Stock conc. in kit | Conc. in assay |
|---|---|---|---|
| Enzyme | hTDO | 90 μM in storage buffer | 50 nM |
| Substrate | L-Tryptophan | 0.8 mM in MilliQ | 0.2 mM |
| Probe | NFK Green™ | Ready to use | 1x |
| Reference compound | NTRC 0672-0 | 10 mM in 100% DMSO | 10 μM |
| TDO assay buffer | Reaction buffer | Ready to use | – |
Advantages of NFK Green™
Detection
NFK Green™ assays can be read out using a fluorescence reader with monochromators or filters that can handle excitation at 400 nm and emission detection at 510 nm. PerkinElmer Envision® multilabel plate readers with 400/25 (excitation; M400w) and 510/20 (emission; M510w) filters and the D425/D490 dual mirror have already been successfully used to measure the signal of NFK Green™ assays in 384- and 1536-well format.
Assay Flowchart
