Recombinant Mouse TDO2 His-tag Protein, CF Summary
with a C terminal 6-His tag
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 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.
- Assay Buffer: 50 mM MES, pH 6.5
- 0.405 M Tris, pH 8.0
- Recombinant Mouse TDO2 (rmTDO2) (Catalog # 10001-TD)
- 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 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 Substrate Mixtures.
a. Dilute L-Tryptophan to 8 mM in Assay Buffer
b. Dilute Ascorbic acid to 80 mM in 0.405 M Tris, pH 8.0.
c. Prepare a mixture of 9000 Units/mL catalase, and 40 µM Methylene Blue in Assay Buffer.
d. Mix equal volumes of 1b and 1c for final concentrations of 40 mM Ascorbic Acid, 4500 units/mL Catalase, and 20 µM Methylene Blue.
- Dilute rmTDO2 to 40 ng/µL in Assay Buffer.
- Load 25 µL of 40 ng/µL rmTDO2 to clear plate, and start the reaction by adding 25 µL of 8 mM L-Tryptophan followed by 50 µL of Mixture 1d. Include a Substrate Blank containing 25 µL Assay Buffer, 25 µL 8 mM L-Tryptophan and 50 µL of Mixture 1d.
- Read plate 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)|
**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:
- rmTDO2: 1.0 µg
- Ascorbic Acid: 20 mM
- L-Tryptophan: 2 mM
- Catalase: 225 units
- Methylene Blue: 10 µM
Recombinant Mouse TDO2 (Catalog # 10001-TD) is measured by its ability to oxidize L-tryptophan to N-formyl-kynurenine.
1 μg/lane of Recombinant Mouse TDO2 was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silver staining, showing bands at 39 kDa and 75 kDa under R conditions.
Tryptophan 2,3-dioxygenase (TDO2), a heme-containing cytosolic dioxygenase, forms a homo-tetrameric active molecule of approximately 190 kDa composed of 48 kDa monomers (1, 2). Mouse TDO2 shares 89% aa sequence identity with human TDO2. TDO2 is one of three proteins 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 (3). TDO2 is a cytosolic protein typically localized to the liver and brain, unlike the more ubiquitously expressed indoleamine 2,3-dioxygenase (IDO), yet it is responsible for ~90% of the primary route of catabolism of tryptophan through the KP (3). TDO2 is upregulated in extrahepatic tumors (4-6) and is consequently a target in cancer immunotherapy (7). TDO2 is a therapeutic target in brain disease such as schizophrenia, Alzheimers disease, multiple sclerosis and glioma (8-11) due to its role in the regulation of levels of critical biologically active downstream KP metabolites (3). Polymorphisms in the TDO2 gene have been implicated for a role in behavioural responses and autism (12,13).
- Lewis-Ballester, A. et al. (2016) Sci. Rep. 6:35169.
- Rafice, S.A. et al. (2009) Biochem. Soc. Trans. 37:408.
- Badawy, A. (2017) Int J. Tryptophan Res. 10:1.
- Pilotte, L. et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:2497.
- D'Amato, N.C. et al. (2015) Cancer Res. 75:4651.
- Yu, C.P et al. (2017) Med. Oncol. 34:73.
- Platten, et al. (2015) Front. Immunol. 5:673.
- Breda, C. et al. (2016) Proc. Natl. Acad. Sci. U.S.A. 113:5435
- Yu, C.P. et al. (2016) Metab. Brain Dis. 31:737.
- Lanz. T.V. et al. (2017) Sci. Rep. 7:41271.
- Reus, G.Z. et al. (2018) Prog. Neuropsychopharmacol. Biol. Psychiatry 81:55.
- Soichot, M. et al. (2013) Alcohol Alcohol 48:415.
- Nabi, R. et al. (2004) Am. J. Med. Genet. B. Neuropsychiatr. Genet. 125B:63.
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