Thioredoxin Reductase (TrxR) Activity Assay Kit (DTNB, Micro Method)

Cat. No.: S1507969

GRADE & PURITY BioReagent

Descripción general

Thioredoxin Reductase (TrxR) is an NADPH-dependent dimeric selenoenzyme containing an FAD domain, belonging to the pyridine nucleotide-disulfide oxidoreductase family. Together with thioredoxin and NADPH, it constitutes the thioredoxin system. TrxR activity is similar to that of GR (glutathione reductase), catalyzing the reduction of GSSG to GSH, making it one of the key enzymes in the glutathione redox cycle.

Detection Principle

TrxR catalyzes the reduction of DTNB by NADPH to generate TNB and NADP⁺. TNB has a characteristic absorption peak at 412 nm. However, reduced glutathione (GSH) can also react with DTNB to produce TNB. The original reduced glutathione in the sample is inhibited using 2-vinylpyridine. By measuring the rate of increase in TNB at 412 nm, TrxR activity can be calculated.

Applicable Samples: Animal tissues, cells, bacteria, serum (plasma)

Please check the quantity of each component before the experiment.

An additional 10% of each component is provided based on the specification for preparing standard curves or preliminary experiments.

Reagents, consumables and Equipments not provided

• Microplate reader (capable of measuring absorbance at 412 nm)

• 96-well microplate (standard transparent plate)

• Deionized water/PBS (for sample dilution/washing)

• Homogenizer (for tissue samples), Constant temperature water bath, Low-temperature centrifuge, Adjustable pipettes and tips (using multichannel pipettes is recommended for efficiency when handling large numbers of samples)

Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Assay Buffer	Ready-to-use, equilibrate to room temperature before use.	Store at 4°C.
Working Chromogen	Prepare immediately before use. Dissolve Chromogen in 3 mL deionized water.	Unused reagent can be stored at 4°C protected from light for up to 2 weeks. Chromogen is somewhat irritating; personal protection is recommended.
Working TrxR Cofactor	Prepare immediately before use. Dissolve TrxR Cofactor in 5 mL deionized water.	Unused reagent can be aliquoted and stored at -20°C protected from light for 2 weeks. Avoid repeated freeze-thaw cycles.
Inhibitor	Ready-to-use, equilibrate to room temperature before use.	Store at -20°C protected from light. Inhibitor is somewhat irritating; personal protection is recommended.

2. Sample Preparation

*Note: All sample processing should be performed on ice. Enzyme activity must be measured on the same day. Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month. For measuring TrxR activity in cells, the cell number should be between 3-5×10⁶. When extracting TrxR from cells, homogenize or sonicate in Assay Buffer; do not use cell lysis buffer.*

2.1 Animal Tissue

Weigh approximately 0.1 g of tissue, add 1 mL of pre-cooled Assay Buffer, and homogenize in an ice bath. Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.

2.2 Cells or Bacteria

Collect 5×10⁶ cells or bacteria, wash with pre-cooled PBS, and centrifuge at 800 g for 2 minutes. Discard the supernatant. Add 1 mL of Assay Buffer and disrupt by sonication on ice. Sonicate for 5 minutes (power 20% or 200 W, pulse 3 seconds, interval 7 seconds, repeat 30 times). Centrifuge at 4°C, 10,000 g for 10 minutes. Collect the supernatant and keep it on ice for assay.

2.3 Serum (Plasma)

Assay directly.

3. Experimental Steps

*Note: For mammalian tissues and blood products, TrxR activity usually needs to be diluted about 5-fold with deionized water. The assay procedure should be performed swiftly. Since the Assay Buffer contains a certain concentration of protein (~0.1 mg/mL), this background protein content must be subtracted when determining sample protein concentration.*

3.1 Microplate Reader Preparation

Preheat for at least 30 minutes and set the wavelength to 412 nm. Incubate the Assay Buffer at 25°C (for general species) or 37°C (for mammals) for 30 minutes.

3.2 Assay System Setup

Only one blank well is needed. Perform the following steps in a 96-well plate:

Reagent	Blank Well (µL)	Test Well (µL)
Assay Buffer	20	0
Sample	0	20
Inhibitor	1	1

Mix well and incubate at 25°C (general species) or 37°C (mammals) for 30 min.

Assay Buffer	140	140
Working Chromogen	20	20
Working TrxR Cofactor	20	20

3.3 Absorbance Measurement

Mix rapidly immediately after adding all reagents. Measure the absorbance at 412 nm at the 10-second mark, recorded as A₁. Incubate the plate at 25°C (general species) or 37°C (mammals) for 5 minutes. Measure the absorbance at the 5-minute 10-second mark, recorded as A₂. Record values for the test well as A_Test and for the blank well as A_Blank.

4. Calculation of Results

Both the derived and simplified calculation formulas provided below are completely equivalent.

4.1 Data Processing

Calculate ΔA = A₂ - A₁, ΔA_Test = A₂_Test - A₁_Test, ΔA_Blank = A₂_Blank - A₁_Blank. Then calculate ΔΔA = ΔA_Test - ΔA_Blank.

4.2 Sample TrxR Activity Calculation

(1) Based on sample protein concentration

Unit definition: One unit (U) is the amount of enzyme that catalyzes the reduction of 1 µmol of DTNB per minute per mg of protein at 25°C or 37°C.

TrxR (U/mg prot) = ΔΔA ÷ (ε × d) × V_{Total reaction} ÷ (Cpr × V_Sample) ÷ T = 0.294 × ΔΔA ÷ Cpr

(2) Based on sample fresh weight

Unit definition: One unit (U) is the amount of enzyme that catalyzes the reduction of 1 µmol of DTNB per minute per gram of sample at 25°C or 37°C.

TrxR (U/g) = ΔΔA ÷ (ε × d) × V_{Total reaction} ÷ (V_Sample ÷ V_{Total extract} × W) ÷ T = 0.294 × ΔΔA ÷ W

(3) Based on cell or bacterial density

Unit definition: One unit (U) is the amount of enzyme that catalyzes the reduction of 1 µmol of DTNB per minute per 10⁴ cells or bacteria at 25°C or 37°C.

TrxR (U/10⁴) = ΔΔA ÷ (ε × d) × V_{Total reaction} ÷ (N × V_Sample ÷ V_{Total extract}) ÷ T = 0.294 × ΔΔA ÷ N

(4) Based on sample volume

Unit definition: One unit (U) is the amount of enzyme that catalyzes the reduction of 1 µmol of DTNB per minute per milliliter of liquid at 25°C or 37°C.

TrxR (U/mL) = ΔΔA ÷ (ε × d) × V_{Total reaction} ÷ V_Sample ÷ T = 0.294 × ΔΔA

Parameter Description:

ε: Molar extinction coefficient of the product, 0.0136 L/µmol/cm

d: Light path of the 96-well plate, 0.5 cm

V_{Total reaction}: Total reaction volume, 200 µL = 2×10⁻⁴ L

Cpr: Protein concentration of the supernatant, mg/mL

W: Sample mass, g

V_Sample: Volume of supernatant added to the reaction system, 20 µL = 0.02 mL

V_{Total extract}: Volume of extraction buffer, 1 mL

T: Reaction time, 5 min

N: Number of cells or bacteria, in units of 10⁴ (e.g., for 5×10⁶ cells, N=500).

5. Example Results

Example 1: 0.1 g of mouse liver was processed and assayed in a 96-well plate. ΔA_Blank = A₂ - A₁ = 0, ΔA_Test = A₂_Test - A₁_Test = 1.772 - 1.109 = 0.663. TrxR activity calculated based on sample weight: TrxR (U/g) = 0.294 × 0.663 ÷ 0.1 = 1.949 U/g.

Example 2: 0.1 g of mouse kidney was processed and assayed in a 96-well plate. ΔA_Blank = A₂ - A₁ = 0, ΔA_Test = A₂_Test - A₁_Test = 1.494 - 0.954 = 0.540. TrxR activity calculated based on sample weight: TrxR (U/g) = 0.294 × 0.540 ÷ 0.1 = 1.588 U/g.

Notes

1. Before formal testing, it is recommended to perform a preliminary experiment with 2-3 samples expected to show significant differences, ensuring the absorbance change is linear within 5 minutes.

2. For tissue and cell samples, results can be normalized by measuring protein concentration. We recommend using Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-use BCA Protein Quantification Kit.

3. This kit is compatible with spectrophotometer detection. Adjust the reagent preparation amounts proportionally according to the spectrophotometer's requirements.

4. Biochemical reagents are generally irritating and may have biological toxicity. For your safety and health, maintain biosafety precautions throughout the experiment: wear a lab coat, mask, gloves, head cover, etc., and perform experiments in a fume hood or biosafety cabinet.

5. This product is for scientific research use only and is not intended for clinical diagnosis.

FAQ

Q: What should I do if the sample ΔΔA obtained is too high or too low?

A: If ΔΔA is less than 0.02, you can appropriately increase the sample amount. If ΔΔA is greater than 1.0, further dilute the sample with Assay Buffer, or reduce the amount of sample used for extraction, and repeat the assay.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light,Store at -20°C

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

Each component has a shelf life of 1 year under corresponding storage conditions. S1507969A: Store at 2-8℃ long term (12 months). S1507969B: Store at 2-8℃ long term (12 months). Store in the dark. S1507969C, S1507969D: Store at -20℃ long term (12 months).

Contents & Storage

S1507969	Component	48T	96T	Storage
S1507969A	Assay Buffer	70 mL	70 mL×2	2-8℃
S1507969B	Chromogen	1 EA	1 EA	2-8℃. Store in the dark.
S1507969C	TrxR Cofactor	1 EA	1 EA	-20℃. Store in the dark.
S1507969D	Inhibitor	60 μL	120 μL	-20℃. Store in the dark.