详细说明
- 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.
- ActivityMeasured 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. See Activity Assay Protocol on .
- SourceE. coli-derived Ala2-Pro407, with an N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisMet - Predicted Molecular Mass46 kDa
- SDS-PAGE41 kDa, reducing conditions
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.
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- 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 # )
- 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 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.
- rmIDO: 0.40 µg
- Ascorbic Acid: 20 mM
- L-Tryptophan: 200 µM
- Catalase: 225 units
- Methylene Blue: 10 µM
- 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