Recombinant Human IDO Protein, CF

(1 Review)
  
  • Purity
    >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
  • Endotoxin Level
    <1.0 EU per 1 μg of the protein by the LAL method.
  • Activity
    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. See Activity Assay Protocol on www.RnDSystems.com.
  • Source
    E. coli-derived
    MHHHHHHHuman IDO
    (Ala2 - Gly403)
    Accession # P14902
    N-terminusC-terminus
  • Accession #
  • N-terminal Sequence
    Analysis
    Met
  • Structure / Form
    Monomer
  • Predicted Molecular Mass
    46 kDa
  • SDS-PAGE
    42 kDa, reducing conditions
6030-AO
 
Formulation Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.
Assay Procedure
Materials
  • 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 1012 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
Background: Indoleamine 2,3-dioxygenase/IDO
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).
  • References:
    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.
  • Entrez Gene IDs:
    3620 (Human); 15930 (Mouse)
  • Alternate Names:
    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
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 Enzymatic activity in vitro Anonymous 05/14/2016
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Enzymatic activity in vitro Recombinant Human IDO Protein, CF 6030-AO
Enzymatic activity in vitro: Recombinant Human IDO Protein, CF [6030-AO]

Summary

ApplicationEnzymatic activity in vitro
Reason for RatingWorked exactly as anticipated for in vitro enzyme activity assay

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