Recombinant Mouse IDO Protein, CF

Catalog #: 9157-AO Datasheet / COA / SDS

Indoleamine 2,3-dioxygenase

Catalog #	Availability	Size / Price	Qty
9157-AO-010

R&D Systems Recombinant Proteins and Enzymes

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All Indoleamine 2,3-dioxygenase/IDO Proteins and Enzymes

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Recombinant Mouse IDO Protein, CF Summary

Product Specifications

Purity

>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

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 >1,000 pmol/min/μg, as measured under the described conditions.

Source

E. coli-derived mouse Indoleamine 2,3-dioxygenase/IDO protein
Ala2-Pro407, with an N-terminal Met and 6-His tag

Accession #

P28776

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

46 kDa

SDS-PAGE

41 kDa, reducing conditions

Product Datasheets

9157-AO

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

9157-AO

Formulation	Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol.
Shipping	The product is shipped with polar packs. 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, -20 to -70 °C as supplied. 3 months, -20 to -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 Mouse Indoleamine 2,3-dioxygenase/IDO (rmIDO) (Catalog # 9157-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 (Catalog # DY990)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Prepare the Substrate Mixture.
a. Dilute Ascorbic Acid to 80 mM in 0.405 M Tris, pH 8.0.
b. Prepare a mixture of 800 µM L-Tryptophan, 9000 units/mL Catalase, and 40 µM Methylene Blue in Assay Buffer.
c. 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.
Dilute rmIDO to 8 ng/µL in Assay Buffer.
Load 50 µL of 8 ng/µL rmIDO to clear plate, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate Mixture.
Read plate in kinetic mode for 5 minutes at an absorbance of 321 nm.

Specific Activity (pmol/min/µg) =	Adjusted V_max* (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:

rmIDO: 0.40 µg
Ascorbic Acid: 20 mM
L-Tryptophan: 200 µM
Catalase: 225 units
Methylene Blue: 10 µM

Reconstitution Calculator

Background: Indoleamine 2,3-dioxygenase/IDO

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing intracellular dioxygenase that catalyzes the degradation of the essential amino acid L-tryptophan to N formyl kynurenine. This degradation is the first and rate-limiting step of the L-kynurenine pathway (1). IDO expression is induced in macrophages, plasmacytoid dendritic cells (pDC), and microglia by multiple inflammatory stimuli including IFN-g, TNF-a, CpG DNA, HIV-1 Tat protein, microbial infection, or GITR Ligand activation (2-9). It can be expressed by tumor cells, pDC in tumor draining lymph nodes, and MDSC in breast cancer (10-12). IDO expression is critical for the ability of these cells to induce the activation of resting Treg and the support of immune tolerance to apoptotic cell debris (7, 11, 13). These cells also suppress anti-tumor immunity (10, 12), the development of autoimmunity (13), and the replication of Chlamydia, Herpes simplex virus, and measles virus (3, 4, 14). In the absence of IDO function, activation of pDC does not induce immunosuppression but instead triggers IL-6 production and the redirecting of Treg to produce IL-17 (7). Mouse IDO shares 61% and 87% amino acid sequence identity with human and rat IDO, respectively (15).

References

Munn, D.H. and A.L. Mellor (2016) Trends Immunol. 37:193.
Thomas, S.R. et al. (1994) J. Biol. Chem. 269:14457.
Carlin, J.M. et al. (1989) J. Interferon Res. 9:329.
Adams, O. et al. (2004) Microbes Infect. 6:806.
Planes, R. and E. Bahraoui (2013) PLoS One 8:e74551.
O'Connor, J.C. et al. (2009) J. Neurosci. 29:4200.
Baban, B. et al. (2009) J. Immunol. 183:2475.
Grohmann, U. et al. (2007) Nat. Med. 13:579.
Loughman, J.A. and D.A. Hunstad (2012) J. Infect. Dis. 205:1830.
Holmgaard, R.B. et al. (2015) Cell Rep. 13:412.
Sharma, M.D. et al. (2007) J. Clin. Invest. 117:2570.
Yu, J. et al. (2013) J. Immunol. 190:3783.
Ravishankar, B. et al. (2012) Proc. Natl. Acad. Sci. USA 109:3909.
Obojes, K. et al. (2005) J. Virol. 79:7768.
Habara-Ohkubo, A. et al. (1991) Gene 105:221.

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