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.

Catalog # NFK003-1K        1,000 datapoints
Catalog # NFK003-10K    10,000 datapoints

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

Please contact us for custom-based large quantities or to order separate components (protein, NFK Green™ or buffer).

Advantages of NFK Green™

  • Homogenous: easy mix-and-measure format
  • Selective: minimal assay interference due to use of long fluorescence wavelengths

  • Safe: mild reaction conditions
  • Robust: Z’-factor of 0.8 and validated in high-throughput screening

  • Flexible: applicable in multiple formats (up to 1,536-well plates) and in the presence of DMSO

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

Reference

Seegers et al. (2014) High-throughput fluorescence-based screening assays for tryptophan-catabolizing enzymes. Journal of Biomolecular Screening, 19 (9):1266-1274.

Interested in other NFK Green™ assay kits?

Please visit our NFK Green™ product page.

Questions about our NFK Green™ products?

Please contact us.