Thioredoxin Reductase (TrxR) is an oxidoreductase in the selenoprotein group. Thioredoxin Reductase is important in regulating multiple cell redox signaling pathways, cell growth, gene transcription, and is vital for development. Impaired thioredoxin reductase activity promotes oxidative stress–induced mitochondrial dysfunction, triggering cell death and the development of cardiomyopathy and neurodegeneration. Elevated thioredoxin reductase levels have been exhibited in several cancer cells allowing it to withstand excess oxidation caused by ionization radiation which leads to resistance to chemotherapy. Therefore, monitoring thioredoxin reductase activity is crucial in assessing cellular health and response to oxidative stress.

Thioredoxin Reductase Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring thioredoxin reductase activity in plasma, serum, cell lysates, and other biological liquid samples. In this assay, thioredoxin reductase catalyzes the reduction of 5, 5’-dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB^2-), which generates a strong yellow color at 412 nm. Since in crude biological samples other enzymes can also reduce DTNB, therefore, thioredoxin reductase specific inhibitor is utilized to determine TrxR specific activity. The difference between the two results is the DTNB reduction due to TrxR activity.

• Microplate reader capable of measuring absorbance at 412 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• MilliQ water or another type of double distilled water (ddH₂O)
• Microplate sealer or plastic wrap

1. Add 50 µL of each sample, or positive control to the 96-well microtiter plate.
2. Each sample replicate requires two paired wells, in one sample well, add 10 µL of the inhibitor. In the other sample well, add 10 µL of assay buffer.
3. Add 100 µL of each standard to the 96-well microtiter plate.
4. Initiate the reaction by adding 40 µL Substrate Mix to each well. Carefully shake the plate for a few seconds to mix well.
5. For kinetic reading: Immediately start measuring absorbance at 412 nm continuously at 25°C and record data every minute for 20 minutes, protect from light. The standard curve can be read in end-point mode.