Key features and details
- Assay type: Enzyme activity
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 40 min
- Sample type: Cell culture extracts, Other biological fluids, Tissue Extracts, Urine
Product nameThioredoxin Reductase Assay Kit (Colorimetric)
See all Thioredoxin reductase kits
Sample typeUrine, Cell culture extracts, Other biological fluids, Tissue Extracts
Assay typeEnzyme activity
Assay time0h 40m
Thioredoxin Reductase Assay Kit (ab83463) is a specific assay for detecting Thioredoxin Reductase (TrxR) activity.
In the thioredoxin reductase assay protocol, TrxR catalyzes the reduction of DTNB to TNB2- in the presence of NADPH, which generates a strong yellow color (ODmax = 412 nm).
Other enzymes present in crude biological samples such as glutathione reductase and glutathione peroxidase can also reduce DTNB. In order to measure TrxR-only activity, a TrxR specific inhibitor is used in a separate reaction to determine TrxR specific activity. The difference between total DTNB reduction in the sample and DTNM reduction in the sample in presence of TrxR inhibitor is the value of specific TrxR activity in the sample.
Thioredoxin reductase assay protocol summary:
- add samples and standards to wells
- add reaction mix
- analyze with a microplate reader over 20 min
Thioredoxin reductase (TrxR, EC 126.96.36.199) is a ubiquitous mammalian enzyme that catalyzes the NADPH-dependent reduction of the redox protein thioredoxin, as well as of other endogenous and exogenous compounds such as selenite, lipid hydroperoxides and hydrogen peroxide.
Storage instructionsStore at -20°C. Please refer to protocols.
Components Identifier 100 tests DTNB Red 1 vial NADPH Blue 1 vial TNB Standard Brown 1 vial TrxR Assay Buffer WM 1 x 25ml TrxR Inhibitor Clear 1 vial TrxR Positive Control Green 1 vial
RelevanceThioredoxin reductase (TrxR) (EC 188.8.131.52) is a ubiquitous enzyme which is involved in many cellular processes such as cell growth, p53 activity, and protection against oxidation stress, etc. The mammalian TrxR reduces thioredoxins as well as non-disulfide substrates such as selenite, lipoic acids, lipid hydroperoxides, and hydrogen peroxide.
Cellular localizationTXNRD1: Cytoplasmic. TXNRD2: Cytoplasmic. Nuclear. Microsome. Endoplasmic reticulum. TXNRD3: Mitochondrial.
- EC 184.108.40.206
- gene associated with retinoic and interferon-induced mortality 12 protein
- Gene associated with retinoic and interferon-induced mortality 12 protein (GRIM-12)
Activity of endogenous Brugia thioredoxin reductase from soluble worm lysates following incubation with 1% DMSO or 0.3 μM, 0.1 μM, or 0.03 μM of auranofin in vitro. Percentages indicate the percent activity of TrxR compared to DMSO controls. Thioredoxin reductase activity was significantly reduced (p < 0.05) to 15%, 33% and 69% of endogenous activity, respectively, compared to the activity in DMSO-treated worms.
Thioredoxin reductase activity of worm lysates was assayed using female B. malayi treated in vitro with either 0.3 μM, 0.1 μM, or 0.03 μM auranofin or 1% DMSO. After 5 hours of treatment, worm motility was measured using the Worminator, and then worms (24 in each group) were pooled, washed three times in PBS, and lysed by douncing in a glass homogenizer in assay buffer (ab83463) with 1 mM PMSF. The crude lysates were centrifuged at 10,000 rcf for 15 minutes at 4°C to pellet insoluble material. The total protein concentrations of soluble lysates were measured using the Bradford assay. The soluble lysates were incubated for 20 minutes in assay buffer or assay buffer with a proprietary thioredoxin reductase specific inhibitor before adding a specific substrate, DTNB (5, 5′-dithiobis (2-nitrobenzoic) acid), and measuring activity at 20 second intervals for 40 minutes using the SpectraMax Plus Microplate Reader (Molecular Devices, Sunnyvale, CA) at λ = 412 nm. Lysates were tested in duplicate. TrxR activity was calculated based on the linear amount of TNB produced per minute per mg of total protein and adjusted for background activity from enzymes other than TrxR in the lysates.
Thioredoxin reductase measured in mouse tissue lysates showing activity (mU) per mg protein of sample tested
Thioredoxin reductase measured in cell lysates showing activity (mU) per 1 mln of cells tested
Standard curve (colourimetric) : mean of duplicates (+/-SD) with background readings substracted
Thioredoxin reductase Kinetic Data using ab83463.
ab83463 has been referenced in 14 publications.
- Danzl K et al. Early inhibition of endothelial retinoid uptake upon myocardial infarction restores cardiac function and prevents cell, tissue, and animal death. J Mol Cell Cardiol 126:105-117 (2019). PubMed: 30472251
- Elie BT et al. Bimetallic titanocene-gold phosphane complexes inhibit invasion, metastasis, and angiogenesis-associated signaling molecules in renal cancer. Eur J Med Chem 161:310-322 (2019). PubMed: 30368130
- Ogiwara H et al. Targeting the Vulnerability of Glutathione Metabolism in ARID1A-Deficient Cancers. Cancer Cell 35:177-190.e8 (2019). PubMed: 30686770
- Elie BT et al. A heterometallic ruthenium-gold complex displays antiproliferative, antimigratory, and antiangiogenic properties and inhibits metastasis and angiogenesis-associated proteases in renal cancer. J Biol Inorg Chem 23:399-411 (2018). PubMed: 29508136
- Maryam A et al. Proscillaridin A Promotes Oxidative Stress and ER Stress, Inhibits STAT3 Activation, and Induces Apoptosis in A549 Lung Adenocarcinoma Cells. Oxid Med Cell Longev 2018:3853409 (2018). PubMed: 29576846