Recombinant Human Thioredoxin-1 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Thioredoxin-1 Protein, CF Summary

• Details of Functionality: Measured by its ability to catalyze the reduction of insulin. The reaction leads to precipitation, which can be measured by absorbance at 650 nm. The specific activity is >10 A₆₅₀/min/mg, as measured under the described conditions.
• Source: E. coli-derived human Thioredoxin-1 protein
  Val2-Val105
• Accession #: P10599
• N-terminal Sequence: Val2
• Protein/Peptide Type: Recombinant Enzymes
• Gene: TXN
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 12 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 12 kDa, reducing conditions

Packaging, Storage & Formulations

• 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS, EDTA and DTT.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Reconstitution Instructions: Reconstitute at 1000 μg/mL in sterile PBS.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, 250 mM NaCl, 2 mM EDTA, pH 6.5
  • Recombinant Human Thioredoxin‑1 (rhTRX-1) (Catalog # 1970-TX)
  • Human Insulin (Sigma, Catalog # I-9278)
  • Dithiothreitol (DTT) (Sigma, Catalog # D-0632), 1 M stock in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: Spectramax Plus by Molecular Devices) or equivalent
  1. Dilute rhTRX-1 to 20 µM in Assay Buffer.
  2. Dilute hInsulin to 520 µM in Assay Buffer.
  3. Dilute DTT to 55 mM in Assay Buffer.
  4. Load into a clear plate 50 µL of Assay Buffer, 25 µL of 20 µM rhTRX-1, and 25 µL of 520 µM hInsulin. For a Substrate Blank, load 75 µL of Assay Buffer and 25 µL of 520 μM hInsulin. Start the reaction by adding 10 µL of 55 mM DTT to all wells.
  5. Read at Abs₆₅₀ in kinetic mode for 15 minutes. Use the linear portion of the reaction to determine specific activity.
  6. Calculate specific activity:

  Specific Activity (Abs/min/mg) = (Adjusted Vmax* (Abs/min)**) / (amount of enzyme (mg))

  *Adjusted for Substrate Blank
  **Note: the output of many spectrophotometers in kinetic mode is in mOD

  Per Well:
  • rhTRX-1: 0.00585 mg
  • hInsulin: 118 µM
  • DTT: 5 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Thioredoxin-1 Protein, CF

• Thioredoxin1
• Thioredoxin-1
• TRDX;TRX;TRX1;Trx80;TXN
• Trx1
• TXN
• TXN1

Background

Thioredoxins (Trxs) are a group of small ubiquitous proteins in all living cells that are key regulators of cellular redox balance (1, 2). The mammalian Trx family has three members. The Trx-1, which is a secreted and cellular protein, the mitochondria-specific Trx-2, and the Trx-like cytosolic protein p32TrxL (3‑5). The active site of mammalian Trxs contains two cysteines in the conserved sequence -Y-C-G-P-C-K-. In Trx-1 the conserved cysteine residues are in positions 32 and 35, respectively. Trxs exist either in a reduced or in an oxidized state when the two cysteines at the active site form an intramolecular disulfide bridge. NADPH and the flavoprotein thioredoxin reductase can convert the oxidized Trx into the reduced Trx. Trx-1 is the only extracellular occurring thioredoxin, and is secreted by lymphocytes, hepatocytes, fibroblasts, and several tumor cells. Plasma concentrations of Trx-1 are up to 6 nM (6). In cells, Trx-1 is localized predominantly in the cytoplasm. Small amounts have been detected in the nucleus and in association with the outside surface of the cells. Expression of Trx-1 is increased under various stress conditions such as hypoxia, elevated hydrogen peroxide concentrations, photochemical oxidative stress, and viral and bacterial infections. Biological functions of Trx-1 include growth factor activity, antioxidant properties, a cofactor that provides reducing equivalents, and transcriptional regulation (1, 2). The synovial tissue of rheumatoid arthritis patients produces increased levels of Trx-1 under oxidative stress conditions, and a correlation exists between the plasma levels of Trx-1 and the severity of the disease, making Trx-1 a biomarker for this pathological condition (7, 8).

1. Holmgren, A. (1985) Annu. Rev. Biochem. 54:237.
2. Powis, G. and W.R. Monfort (2001) Annu. Rev. Pharm. Toxicol. 41:269.
3. Deiss, L.P. and A. Kimchi (1991) Science 252:117.
4. Spyrou, G. et al. (1997) J. Biol. Chem. 272:2936.
5. Miranda-Vizuete, A. et al. (1998) Biochem. Biophys. Res. Commun. 243:284.
6. Nakamura, H. et al. (1997) Annu. Rev. Immunol. 15:147.
7. Mourice, M.M. et al. (1999) Arthritis Rheum. 42:2430.
8. Jikimoto, T. et al. (2001) Mol. Immunol. 38:765.

Publications for Thioredoxin-1 (1970-TX)(4)

Publications using 1970-TX

• Gram Negative Bacterial Inflammation Ameliorated by the Plasma Protein Beta 2-Glycoprotein I Sci Rep, 2016-09-27;6(0):33656. 2016-09-27 [PMID: 27670000] (Enzyme Assay, Human)
• Lundberg M, Curbo S, Reiser K, Masterman T, Braesch-Andersen S, Arestrom I, Ahlborg N Methodological aspects of ELISA analysis of thioredoxin 1 in human plasma and cerebrospinal fluid. PLoS ONE, 2014-07-30;9(7):e103554. 2014-07-30 [PMID: 25075746] (ELISA (Standard), Human)
• Kodama A, Watanabe H, Tanaka R, Tanaka H, Chuang V, Miyamoto Y, Wu Q, Endo M, Hamasaki K, Ishima Y, Fukagawa M, Otagiri M, Maruyama T A human serum albumin-thioredoxin fusion protein prevents experimental contrast-induced nephropathy. Kidney Int, 2013-01-02;83(3):446-54. 2013-01-02 [PMID: 23283135] (Bioassay, Rat)
• Passam FH, Rahgozar S, Qi M, Raftery MJ, Wong JW, Tanaka K, Ioannou Y, Zhang JY, Gemmell R, Qi JC, Giannakopoulos B, Hughes WE, Hogg PJ, Krilis SA Redox control of beta2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1. J. Thromb. Haemost., 2010-08-01;8(8):1754-62. 2010-08-01 [PMID: 20979592] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: TXN
• Uniprot:
  • P10599()