Recombinant Human IDO Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human IDO Protein, CF Summary

• Additional Information: Indoleamine 2,3-dioxygenase
• Details of Functionality: Measured by its ability to oxidize L-tryptophan to N-formyl-kynurenine. The specific activity is >500 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Indoleamine 2,3-dioxygenase/IDO protein
  

  M	HHHHHH	Human IDO (Ala2-Gly403) Accession # P14902
  N-terminus		C-terminus

• Accession #: P14902
• N-terminal Sequence: Met
• Structure / Form: Monomer
• Protein/Peptide Type: Recombinant Enzymes
• Gene: IDO1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 46 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 42 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, pH 6.5
  • 0.405 M Tris, pH 8.0
  • Recombinant Human Indoleamine 2,3‑dioxygenase/IDO (rhIDO) (Catalog # 6030-AO)
  • Ascorbic Acid (Sigma, Catalog # 255564), 500 mM stock in deionized water
  • L-Tryptophan (Sigma, Catalog # T0254), 10 mM stock in deionized water
  • Catalase (Sigma, Catalog # C30), 100,000 units/mL stock in Assay Buffer
  • Methylene Blue (Sigma, Catalog # 28514), 10 mM stock in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare the Substrate Mixture.
  1. Dilute Ascorbic Acid to 80 mM in 0.405 M Tris, pH 8.0.
  2. Prepare a mixture of 800 μM L-Tryptophan, 9000 units/mL catalase, and 40 μM Methyene Blue in Assay Buffer.
  3. Mix equal volumes of 1a and 1b for final concentrations of 40 mM Ascorbic Acid, 400 μM L-Tryptophan, 4500 units/mL Catalase, and 20 μM Methylene Blue.
  2. Dilute rhIDO to 16 ng/μL in Assay Buffer.
  3. Load into a plate 50 μL of 16 ng/μL rhIDO, and start the reaction by adding 50 μL of Substrate Mixture.
  4. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
  5. Read at 321 nm in kinetic mode for 5 minutes.

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 3750 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD. Per Well:
  • rhIDO: 0.800 µg
  • Ascorbic Acid: 20 mM
  • L-Tryptophan: 200 μM
  • Catalase: 225 units
  • Methylene Blue: 10 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human IDO Protein, CF

• 3dioxygenase
• EC 1.13.11.52
• IDO
• IDO1
• IDOIDO-1
• INDO
• INDOindole 2,3-dioxygenase
• Indoleamine 2
• indoleamine 2,3-dioxygenase 1
• Indoleamine 2,3-dioxygenase
• indoleamine-pyrrole 2,3 dioxygenase
• Indoleamine-pyrrole 2,3-dioxygenase

Background

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing intracellular dioxygenase catalyzing the degradation of the essential amino acid L-tryptophan to N‑formyl‑kynurenine (1). This degradation is the first and rate-limiting step of the L-kynurenine pathway (2). IDO is widely expressed in dendritic cells, macrophages, microglia, eosinophils, fibroblasts, endothelial cells, and most tumor cells. In immune cells, its expression is mainly induced by cytokines such as IFN‑ gamma , IFN‑ alpha , IFN‑ beta , and IL‑10. IDO has an antimicrobial function due to its decreasing the availability of the essential amino acid tryptophan in inflammatory environments (3). Recent studies have demonstrated that IDO induces immunosuppression during infection, pregnancy, transplantation, autoimmunity, and neoplasia (3‑5).

1. Lewis-Ballester, A. et al. (2009) Proc. Natl. Acad. Sci. USA. 106:17371.
2. Costantino, G. (2009) Expert Opin. Ther. Targets 13:247.
3. Xu, H. et al. (2008) Immunol. Lett. 121:1.
4. Lob, S. et al. (2009) Nat. Rev. Cancer 9:445.
5. Curti, A. et al. (2009) Blood 113:2394.

Publications for Indoleamine 2,3-dioxygenase/IDO (6030-AO)(3)

Publications using 6030-AO

• DW Donley, M Realing, JP Gigley, JH Fox Iron activates microglia and directly stimulates indoleamine-2,3-dioxygenase activity in the N171-82Q mouse model of Huntington&#039;s disease PLoS ONE, 2021-05-14;16(5):e0250606. 2021-05-14 [PMID: 33989290] (Bioassay, Human)
• H Jonescheit, HH Oberg, D Gonnermann, M Hermes, V Sulaj, C Peters, D Kabelitz, D Wesch Influence of Indoleamine-2,3-Dioxygenase and Its Metabolite Kynurenine on gammadelta T Cell Cytotoxicity against Ductal Pancreatic Adenocarcinoma Cells Cells, 2020-05-06;9(5):. 2020-05-06 [PMID: 32384638] (N/A)
• E Bracho-San, A Hassanzade, MA Brusko, MA Wallet, BG Keselowsky Dendritic Cells Treated with Exogenous Indoleamine 2,3-Dioxygenase Maintain an Immature Phenotype and Suppress Antigen-specific T cell Proliferation J Immunol Regen Med, 2019-02-10;5(0):. 2019-02-10 [PMID: 31788580] (Bioassay, Human, Mouse)

Bioinformatics

• Gene Symbol: IDO1
• Uniprot:
  • P14902()