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Thioredoxin Reductase Assay Kit (ab83463) is a specific assay for detecting Thioredoxin Reductase (TrxR) activity in various samples. TrxR catalyzes the reduction of DTNB (5, 5'-dithiobis (2-nitrobenzoic) acid) to TNB2- (5-thio-2-nitrobenzoic acid) in 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.
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Thioredoxin reductase (TrxR, EC 220.127.116.11) 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. TrxR is the only enzyme known to catalyze the reduction of thioredoxin in order to form reduced disulfide bounds in cells, and it is likely that also plays a role in protection against oxidant injury, cell growth and transformation.
|TNB Standard||Brown||1 vial|
|TrxR Assay Buffer||WM||1 x 25ml|
|TrxR Inhibitor||Clear||1 vial|
|TrxR Positive Control||Green||1 vial|
Our Abpromise guarantee covers the use of ab83463 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent dilution.|
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
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.
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