Thioredoxin Reductase Assay Kit

(Catalog #K763-100; 100 reactions; Store kit at –20^oC)

1. 
   

   Introduction:

   
   

Thioredoxin reductase (TrxR) (EC 1.8.1.9) 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. BioVision’s Thioredoxin Reductase Assay Kit provides a convenient colorimetric assay for detecting TrxR activity in various samples. In the assay TrxR catalyzes the reduction of 5, 5’-dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB^2-), which generate a strong yellow color (λ_max = 405 nm). Since in crude biological samples other enzymes, such as glutathione reductase and glutathione peroxidase, can also reduce DTNB, therefore, TrxR specific inhibitor is utilized to determine TrxR specific activity. Two assays are performed: the first measurement is of the total DTNB reduction by the sample, and the second one is the DTNB reduction by the sample in the presence of the TrxR specific inhibitor. The difference between the two results is the DTNB reduction by TrxR.

2. 
   

   Kit Contents:

   
   

3. 
   

   Storage and Handling:

   
   

Store the kit at -20^oC, protect from light. Warm Assay Buffer to room temperature before use. Briefly centrifuge vials prior to opening. Read the entire protocol before performing the assay.

4. 
   

   Reagent Reconstitution and General Consideration:

   
   

TNB Standard: Dissolve lyophilized TNB standard into 0.5 ml Assay Buffer to generate 5 mM TNB Standard. The TNB standard solution is stable for 1 week at 4°C or 2 month at -20°C.

DTNB Solution: Dissolve DTNB into 0.9 ml Assay Buffer, sufficient for 100 assays. The DTNB solution is stable for 1 week at 4°C or 2 month at -20°C.

NADPH: Dissolve one vial with 0.22 ml dH₂O; sufficient for 100 assays. The solution is stable for 1 week at 4°C or 2 month at -20°C.

TrxR Positive Control: Dilute 10 µl TrxR with 90 µl Assay Buffer to generate ~0.2 mU/l TrxR; it is stable for 1 day at 4°C or 2 month at -20°C.

TrxR Inhibitor: Dissolve TrxR Inhibitor into 1.2 ml Assay Buffer, sufficient for 100 assays. The TrxR Inhibitor solution is stable for 2 month at -20°C.

Ensure that the Assay Buffer is at room temperature before use. Keep samples, NADPH, TrxR inhibitor, TrxR Positive Control on ice during the assay.

5. 
   

   Glutathione Reductase Activity Assay:

   
   

0. 
   

   TNB Standard Curve:
   
   Add 0, 2, 4, 6, 8, 10 µl of the TNB Standard into 96-well plate in duplicate to generate 0, 10, 20, 30, 40, 50 nmol/well standard. Bring the final volume to 100µl with Assay Buffer.

   
   


1. 
   

   Sample and Positive Control Preparations:
   
   Take 20 mg Tissues or 2x10⁶ Cells and homogenize in 100-200l cold Assay Buffer on ice (It is recommended to add Protease Inhibitor Cocktail (Cat.# K271-500) to the buffer); Centrifuge at 10,000 x g for 15 min at 4°C; Collect the supernatant for assay and store on ice.

   
   

2. 
   

   Serum can be tested directly. Determine the protein concentration of the supernatant using the Bradford Reagent (BioVision Cat # K810-100). Keep samples at -80°C for storage.

   
   


3. 
   

   Assay Procedure: Add 2-50 µl sample or 10 µl TrxR positive control into each well, adjust volume to 50 µl with assay buffer. 2 sets of samples should be tested as with or without TrxR Inhibitor. Add 10 l of TrxR Inhibitor to one set of the sample for testing background enzyme activity, and add 10 l of Assay Buffer to the other set of sample for testing total DTNB reduction, mix well.

   
   


4. 
   

   Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 40 μl Reaction Mix:

   
   

30 μl Assay Buffer

8 μl DTNB Solution

2 μl NADPH

5. 
   

   Add 40 μl of the Reaction Mix to each test sample, mix well. Measure O.D.405 nm at T₁ to get A_1t and A_1I, measure O.D.405 nm again at T2 after incubating the reaction at 25°C for 20 min (The incubate time can vary depend on the sample concentration) to get A_2t and A_2I, protect from light. The O.D. of TNB^2- generated by TrxR is ∆A_{405 nm} = (A_2t – A_1t) - (A_2I-A_1I).

   
   

Note: It is essential to read A_1t, A_1I, A_2t and A_2I’ in the reaction linear range. It will be more accurate if you read the reaction kinetics. Then choose A_1t, A_1I, A_2t and A_2I in the reaction linear range.

6. 
   

   Calculation: Plot the TNB standard Curve. Apply the ∆A_405nm to the TNB standard curve to get B nmol of TNB (TNB amount generated between T1 and T2 in the reaction wells).

   
   

B

TrxR Activity = ^{___________________} X Sample Dilution Factor = nmol/min/ml = mU/mL

(T2-T1) x V

Where: B is the TNB amount from TNB standard Curve (in nmol).

T1 is the time of the first reading (A_1t and A_1I) (in min).

T2 is the time of the second reading (A_2t and A_2I) (in min).

V is the pretreated sample volume added into the reaction well (in ml).

TrxR Unit Definition: One unit of TrxR is the amount of enzyme that generates1.0 µmol of TNB per minute at 25°C. The oxidation of 1 mole of NADPH to NADP will generate 2 mole TNB finally, therefore, 1 TNB unit equals 0.5 NADP unit.