Recombinant Human IDO2 Protein, CF Summary
Glu15-Gly420, with an N-terminal Met and C-terminal 6-His tag
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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.
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, 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.
- Assay Buffer: 50 mM Tris, 20% Glycerol, pH 7.5
- 0.405 M Tris, pH 8.0
- Recombinant Human IDO2 (rhIDO2) (Catalog # 9967-AO)
- Ascorbic Acid (Sigma, Catalog # 255564), 500 mM stock in deionized water
- L-Tryptophan (Sigma, Catalog # T0254), 40 mM stock in deionized water
- Catalase (Sigma, Catalog # C30), 100,000 units/mL stock diluted in 50 mM MES, pH 6.5
- 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 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, 4500 units/mL Catalase and 20 µM Methylene
- Dilute rhIDO2 to 160 ng/µL in Assay Buffer.
- Load 25 µL of 160 ng/µL of rhIDO-2 to a clear plate, and start the reaction by adding 25 µL of 40 mM L-Tryptophan, followed by 50 µL of Substrate Mixture. Include a Substrate Blank containing 25 µL of Assay Buffer, 25 µL of L-Tryptophan and 50 µL of Substrate Mixture.
- Read in kinetic mode for 5 minutes at an absorbance of 321 nm.
- Calculate specific activity:
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
- rhIDO2: 4.0 μg
- Ascorbic Acid: 20 mM
- L-Tryptophan: 10 mM
- Catalase: 225 units
- Methylene Blue: 10 µM
Recombinant Human IDO2 (Catalog # 9967-AO) is measured by its ability to oxidize L-tryptophan to N-formyl-kynurenine. The activity (orange) is approximately 4-fold greater than the competitor's IDO2 (green).
1 μg/lane of Recombinant Human IDO2 was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silverstaining, showing a band at 41 kDa.
Indoleamine 2,3-dioxygenase (IDO2) is a 47 kDa heme-containing cytosolic dioxygenase. Human IDO2 shares 64% aa sequence identity with mouse IDO2. IDO2 is one of three dioxygenases capable of catalyzing the first and rate-limiting step of the L-kynurenine pathway (KP): oxidative cleavage of the essential amino acid L-tryptophan to form N formyl kynurenine (1). Of these proteins, IDO1 and IDO2 are both related, monomeric enzymes but share only 38% aa sequence identity. The IDO isoforms are not functionally redundant. Although expression of IDO2 has been upregulated in some cancers (2,3), IDO2 expression is generally restricted to the liver, kidney, brain, and certain immune cell types unlike the more ubiquitously expressed indoleamine 2,3-dioxygenase (IDO) (1). Differential inhibition of IDO1 and IDO2 is observed with several molecules (4,5). IDO2 has significantly lower tryptophan catabolic activity than IDO1 and TDO2 (4, 6-8) suggesting it does not play a significant physiological role in the KP. Instead, IDO2 may have an alternative functional role: either non-enzymatic or utilizing a more physiologically relevant substrate (5,7). IDO2 function operates as a pro-inflammatory mediator in autoimmune inflammatory disorders (8, 9-11). It is a candidate for co-therapeutic targeting for treatment in these diseases (10-11).
- Prendergast, G.C. et al. (2014) Front. Immunol. 5:585.
- Witkiewicz, A.K. et al. (2009) J. Am. Coll. Surg. 208:781.
- Lob, S. et al. (2009) Cancer Immunol. Immunother. 58:153.
- Meininger, D. et al. (2011) Biochim. Biophys. Acta. 1814:1947.
- Pantouris, G. et al. (2014) Amino Acids 46:2155.
- Ball, H.J. et al. (2007) Gene 396:203.
- Yuasa, H.J. et al. (2009) Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 153:137.
- Metz, R. et al. (2014) Int. Immunol. 26:357.
- Merlo, L.M.F. et al. (2014) J. Immunol. 192:2082.
- Merlo, L.M. and L. Mandik-Nayak (2016) Clin. Med. Insights Pathol. 9:21.
- Merlo, L.M.F. et al (2017) Clin. Immunol. 179:8.
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