Total Antioxidant Capacity (T-AOC) Assay Kit (ABTS)

Cat. No.: T1507359

GRADE & PURITY BioReagent

Descripción general

  Reactive oxygen species (ROS) mainly include hydroxyl radicals, superoxide radicals, and hydrogen peroxide. ROS are generated during the normal physiological metabolism of cells or tissues; meanwhile, some environmental factors such as ultraviolet (UV) radiation and environmental pollution can also induce ROS production. Once produced, ROS can cause oxidative damage to lipids, proteins, DNA, and other substances in cells, trigger oxidative stress, and subsequently lead to various diseases, including tumors, atherosclerosis, rheumatoid arthritis, diabetes, liver injury, and central nervous system diseases. The body contains a variety of antioxidants, including antioxidant macromolecules, antioxidant small molecules, and enzymes. These antioxidants can scavenge various ROS produced in the body to prevent oxidative stress induced by ROS. The total level of antioxidant macromolecules, antioxidant small molecules, and enzymes in a system reflects the total antioxidant capacity (T-AOC) of that system. Therefore, determining the total antioxidant capacity in various body fluids (such as plasma, serum, urine, and saliva) and lysates (such as those from cells or tissues) holds significant biological importance.

  This kit uses ABTS as a chromogenic reagent and is designed to determine the total antioxidant capacity (T-AOC) of various samples, including body fluids (such as plasma, serum, saliva, and urine), lysates (such as those from cells or tissues), extracts from plants or Chinese herbal medicines, and solutions of various antioxidants. The detection principle of T-AOC determination by the ABTS method is as follows: ABTS is oxidized to green ABTS⁺ under the action of an oxidizing agent; in the presence of antioxidants, the generation of ABTS⁺ is inhibited. The total antioxidant capacity of the sample can be determined and calculated by measuring the absorbance of ABTS⁺ at a wavelength of 734 nm or 405 nm. Trolox is an analog of vitamin E with antioxidant capacity similar to that of vitamin E, and it can be used as a reference for the total antioxidant capacity of other antioxidants. For example, the total antioxidant capacity of Trolox is defined as 1; under the condition of the same concentration, the antioxidant capacity of other substances can be expressed as a multiple relative to that of Trolox. This kit is fast and convenient: absorbance measurement can be performed 3–6 minutes after adding the sample to be tested, and usually 10–20 samples can be tested within 20 minutes. This kit is for research use only and not intended for clinical diagnosis or other purposes.

T1507359	Component	100 T	300 T	1000 T	Storage
T1507359A	ABTS Solution	0.5ml	1.5ml	5ml	-20℃. Avoid freeze/thaw cycle. Store in the dark.
T1507359B	Oxidizing agent	0.5ml	1.5ml	5ml	2-8℃.
T1507359C	Trolox	5mg	5mg	5mg	2-8℃. Store in the dark.

Materials to Prepare by Yourself:

1. Experimental Materials: Plant tissues (e.g., apples, bananas, pears, corn), blood, tissue samples

2. Reagents/Solvents: Distilled water, absolute ethanol, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), etc.

3. Instruments/Equipment: Mortar or homogenizer, centrifuge, centrifuge tubes, microplate reader, 96-well plate

Operating Procedures (For Reference Only):

1. Sample Preparation:

① Plant Samples: Take fresh plant tissues (under normal or specific conditions), rinse and dry them thoroughly, then chop them into pieces and weigh quickly. Homogenize or grind the tissues at a ratio of 0.5 g sample : 4.5 mL distilled water. Centrifuge at 12,000 rpm, 4°C for 5 minutes, and collect the supernatant for later use.

② Plasma, Serum, Saliva, and Urine Samples:Freshly collected samples can be directly tested with this kit, or stored at -80°C for later testing (no significant changes will occur within one month).

Note: EDTA should not be used for anticoagulation during plasma preparation.

③ Tissue Samples:Accurately weigh 50 mg of tissue, add 200 μL pre-cooled PBS, and process via ultrasonication or homogenization to fully disrupt cells and release antioxidants. Centrifuge at 12,000 rpm, 4°C for 5 minutes, and collect the supernatant for later use.

④ Cell Samples:Collect approximately 1 million cells (no accurate counting required; scrape cells directly without trypsin or EDTA digestion). Add 200 μL pre-cooled PBS, and process via ultrasonication or homogenization to fully disrupt cells and release antioxidants. Centrifuge at 12,000 rpm, 4°C for 5 minutes, and collect the supernatant for later use.

⑤ Antioxidant Substances:Dissolve the antioxidant in water or other solvents to a concentration of 0.1–1.5 mM; the solution is then ready for testing.

⑥ If the antioxidant capacity of a sample is relatively high, dilute the sample with distilled water or the corresponding solvent before retesting.

2. Preparation of ABTS Working Solution:

Prepare an appropriate volume of ABTS working solution based on the number of samples to be tested (including the standard curve).

① Mix ABTS solution and oxidizing agent in equal volumes to prepare ABTS stock working solution. Store it at room temperature in the dark for 12–16 hours before use. The prepared ABTS stock working solution is stable for 2–3 days when stored at room temperature in the dark.【For 20 samples to be tested, take 0.1 mL each of ABTS solution and oxidizing agent.】

② Before use, dilute the ABTS stock working solution with absolute ethanol, PBS, or distilled water as required to prepare the ABTS working solution: For water-insoluble samples, use absolute ethanol for dilution; For water-soluble samples, use PBS or distilled water for dilution. The dilution factor of the ABTS stock working solution is approximately 30–55 times.

Note: After subtracting the absorbance of the corresponding blank control (absolute ethanol, PBS, or distilled water) from the absorbance of the ABTS working solution: For a microplate reader (300 μL reaction volume), the absorbance at 734 nm (A₇₃₄) should be 0.5–1.5, and the corresponding absorbance at 405 nm (A₄₀₅) should be 1.3–3.5; For a spectrophotometer (1 cm light path), the A₇₃₄ should be 0.7–1.8, and the corresponding A₄₀₅ should be 1.5–4.0.

3. Preparation of Trolox Solution (10 mM): Add 2 mL of absolute ethanol or dimethyl sulfoxide (DMSO) to 5 mg of Trolox and mix until fully dissolved.

4. Preparation of Trolox Standard Gradients: Dilute the 10 mM Trolox solution with an appropriate solvent to concentrations of 0.05, 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. Use the following solvents for dilution based on sample type: For serum, plasma, saliva, or urine samples: Dilute the standard with distilled water or PBS directly; For tissue or cell samples: Dilute the standard with PBS or the corresponding tissue lysis buffer; For other samples: Dilute the standard with the corresponding sample preparation solvent.

5. Sample Loading: Set up blank wells, standard wells, and test wells according to the table below. Add the solutions to the 96-well plate in sequence, avoid generating air bubbles, and mix gently.【The volume ratio of ABTS working solution to the sample/standard to be tested is 40:1.】

Incubate at room temperature for 3–6 minutes, then perform absorbance measurement. If the absorbance value of a sample is too high, reduce the sample volume or dilute the sample appropriately before retesting. It is recommended to set up duplicate tubes for sample testing.

Substances to Add (μl)	Blank Well	Standard Well	Test Well
ABTS Working Solution	280	280	280
Distilled water	7	-	-
Serial Trolox Standards (No. 1–No. 7)	-	7	-
Test Sample Solution	-	-	7

6. Measurement: Use a microplate reader to measure the absorbance at 405 nm or 734 nm, and record the values as A_Blank、A_Standard、A_{Test .}

Calculations:

Plot a standard curve for total antioxidant capacity (T-AOC), with the serial Trolox standards as the x-axis (abscissa) and the corresponding absorbance values as the y-axis (ordinate). Based on the absorbance value of the sample test tube, calculate the antioxidant capacity of the sample in terms of the equivalent concentration of Trolox standard.

When determining the T-AOC of cell or tissue samples, the protein concentration must be measured simultaneously. The final T-AOC result is usually expressed as the T-AOC per milligram or gram of protein, with units of mmol/mg or mmol/g. Example: If the protein concentration of a lysate sample is 0.15 mg/mL, and its inhibition rate is equivalent to that of 0.3 mM Trolox, the T-AOC of the lysate sample is calculated as 0.3 mM ÷ 0.15 mg/mL = 2 mmol/g.

The antioxidant capacity of plant or Chinese herbal medicine extracts can be expressed as the T-AOC per milligram or gram of the extract’s dry weight, with units of mmol/mg or mmol/g. Example: If the concentration of a sample is 0.1 mg/mL, and its inhibition rate is equivalent to that of 0.5 mM Trolox, the T-AOC of the sample is calculated as 0.5 mM ÷ 0.1 mg/mL = 5 mmol/g.

When various antioxidants are expressed in molar concentration, their measured T-AOC is represented as relative total antioxidant capacity. Example: If the OD value of a 0.2 mM antioxidant is equivalent to that of 1 mM Trolox, its relative total antioxidant capacity is 5 (1 mM ÷ 0.2 mM = 5).

The antioxidant capacity of mixtures such as plasma, serum, saliva, or urine can be directly expressed in the molar concentration of Trolox.

Example 1: If the inhibition rate of a sample is equivalent to that of 0.6 mM Trolox, the T-AOC of the sample is 0.6 mM.

Example 2: If a sample is diluted 5-fold, and its inhibition rate is equivalent to that of 0.5 mM Trolox, the T-AOC of the original sample is 0.5 mM × 5 = 2.5 mM.

Free Radical Scavenging Rate (%) of the Sample = [(A₈ₗₐₙₖ − Aₜₑₛₜ) ÷ A₈ₗₐₙₖ] × 100

Precautions:

1. Experimental materials should be as fresh as possible. The entire sample extraction process is preferably performed at 4°C or on ice. If immediate detection is not possible after sample collection, the samples can be stored at -80°C for later testing (testing must be completed within 1 month).

2. Substances that affect redox reactions (such as DTT and mercaptoethanol) must not be added to the samples. Detergents (such as Tween, Triton, and NP-40) should also not be added.

3. A microplate reader capable of measuring at 734 nm or a spectrophotometer suitable for micro-sample measurement must be used for detection.

4. Trolox solution solidifies at low temperatures; before use, it should be dissolved with the assistance of a water bath at approximately 40°C.

5. If it is difficult to detect the sample at 734 nm or the sample causes interference, try detecting at 405 nm. For cell or tissue samples, detection at 405 nm may cause certain interference to the results due to the inherent absorbance of the samples themselves.

6. ABTS is irritating to the human body. Please operate with caution and take protective measures to avoid direct contact with the body or inhalation.

7. According to literature reports: The total antioxidant capacity (T-AOC) of human serum or plasma is 0.5–2 mM; The T-AOC of saliva is 0.3–1 mM; The T-AOC of urine is 0.2–3 mM; The antioxidant capacity of vitamin C is 1.0; The antioxidant capacity of vitamin E is 1.0; The antioxidant capacity of GSH is 1.3; The T-AOC of fresh orange juice is 2.2.

8. This kit is limited to scientific research use by professional personnel. It must not be used for clinical diagnosis or treatment, nor for food or pharmaceuticals. It must not be stored in ordinary residential areas.

9. For your safety and health, please wear a lab coat and disposable gloves during operation.

10. After opening, the reagents should be used as soon as possible to avoid affecting the results of subsequent experiments.

Appendix: Preparation of the Standard Curve

Operate at room temperature as follows:Dilute 0.25 mL of ABTS stock working solution with 9.75 mL of absolute ethanol at a 1:50 dilution ratio to prepare the ABTS working solution. The absorbance values are A₇₃₄ = 0.692 and A₄₀₅ = 1.445.

Dilute the 10 mM Trolox solution with absolute ethanol to obtain concentrations of 0.05 mM, 0.15 mM, 0.3 mM, 0.6 mM, 0.9 mM, 1 mM, 1.2 mM, and 1.5 mM (note: corrected the possible typo "06" to "0.6" for concentration accuracy).

Take 280 μL of the ABTS working solution, then add 7 μL of distilled water (blank control) or each Trolox standard respectively. Mix well, incubate at room temperature for 6 minutes, and measure the absorbance of each tube at 405 nm using a microplate reader. The absorbance values and standard curve are provided below (for reference only):

Trolox Standard	Absorbance
0	1.500
0.05	1.409
0.15	1.307
0.3	1.207
0.6	0.806
0.9	0.466
1.0	0.380
1.2	0.045
1.5	0.045

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light,Store at -20°C,Avoid repeated freezing and thawing

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

Each component has a shelf life of 1 year under corresponding storage conditions.