Biochemical Assay Kit for Human IDO1 (hIDO1)

NFK Green™ Assay Technology

Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that converts L-tryptophan into NFK, which is the first step in the kynurenine pathway of tryptophan degradation. IDO1 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 IDO1 (hIDO1) biochemical assay kit can be used to determine the potency of IDO1 inhibitors.

Catalog # NFK001-1K        1,000 datapoints
Catalog # NFK001-10K    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 IDO1 inhibitor in the NFK Green™ hIDO1 assay.

Kits contents

Component Description Stock conc. in kit Conc. in assay
Enzyme hIDO1 50 μM in storage buffer 25 nM
Substrate L-Tryptophan 0.4 mM in MilliQ 0.1 mM
Probe NFK Green™ Ready to use 1x
Reference compound NTRC 0820-0 10 mM in 100% DMSO 10 μM
IDO1 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 0.7 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.