Recombinant Mouse TDO2 His-tag Protein, CF

Catalog # Availability Size / Price Qty
10001-TD-020
TDO2 Protein Enzyme Activity
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Recombinant Mouse TDO2 His-tag Protein, CF Summary

Purity
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<0.10 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.
Source
E. coli-derived mouse TDO2 protein
Val18-Phe388
with a C terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Val18
Predicted Molecular Mass
45 kDa
SDS-PAGE
39 & 75 kDa, reducing conditions

Product Datasheets

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.

10001-TD

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.
  • 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 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
  1. 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.
  2. Dilute rmTDO2 to 40 ng/µL in Assay Buffer.
  3. 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.
  4. Read plate in kinetic mode for 5 minutes at an absorbance of 321 nm.
  5. 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. Per Well:
  • rmTDO2: 1.0 µg
  • Ascorbic Acid: 20 mM
  • L-Tryptophan: 2 mM
  • Catalase: 225 units
  • Methylene Blue: 10 µM

Data Images

Enzyme Activity Enzyme Activity View Larger

Recombinant Mouse TDO2 (Catalog # 10001-TD) is measured by its ability to oxidize L-tryptophan to N-formyl-kynurenine.

SDS-PAGE SDS-PAGE View Larger

1 μg/lane of Recombinant Mouse TDO2 was resolved with SDS-PAGE underreducing (R) and non-reducing (NR) conditions and visualized by silver staining,showing bands at 39 kDa and 75 kDa under R conditions.

Reconstitution Calculator

Reconstitution Calculator

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Background: TDO2

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).

References
  1. Lewis-Ballester, A. et al. (2016) Sci. Rep. 6:35169.
  2. Rafice, S.A. et al. (2009) Biochem. Soc. Trans. 37:408.
  3. Badawy, A. (2017) Int J. Tryptophan Res. 10:1.
  4. Pilotte, L. et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:2497.
  5. D'Amato, N.C. et al. (2015) Cancer Res. 75:4651.
  6. Yu, C.P et al. (2017) Med. Oncol. 34:73.
  7. Platten, et al. (2015) Front. Immunol. 5:673.
  8. Breda, C. et al. (2016) Proc. Natl. Acad. Sci. U.S.A. 113:5435
  9. Yu, C.P. et al. (2016) Metab. Brain Dis. 31:737.
  10. Lanz. T.V. et al. (2017) Sci. Rep. 7:41271.
  11. Reus, G.Z. et al. (2018) Prog. Neuropsychopharmacol. Biol. Psychiatry 81:55.
  12. Soichot, M. et al. (2013) Alcohol Alcohol 48:415.
  13. Nabi, R. et al. (2004) Am. J. Med. Genet. B. Neuropsychiatr. Genet. 125B:63.
Long Name
Tryptophan 2,3-dioxygenase
Entrez Gene IDs
6999 (Human); 56720 (Mouse); 64206 (Rat)
Alternate Names
chky; TDO; TDO2; TDOTO; TO; TPH2EC 1.13.11.11; TRPO; TRPOtryptophanase; Tryptamin 2,3-dioxygenase; tryptophan 2,3-dioxygenase; Tryptophan Oxygenase; Tryptophan Pyrrolase; Tryptophanase

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