Product Introduction
ABTS assay is the most widely used indirect detection method for determining the antioxidant capacity of both hydrophilic and lipophilic substances. It is suitable for liquid samples such as plant tissues and red wine. Its principle is as follows: ABTS is oxidized to form stable blue-green cationic radical ABTS⁺, which is soluble in aqueous phase or acidic ethanol medium and exhibits maximum absorbance at 734 nm. After the test substance is added into the ABTS⁺ solution, the antioxidant components contained in the sample react with ABTS⁺ and decolorize the reaction system. The absorbance change is measured at 734 nm, and vitamin C is used as the reference system to quantify the antioxidant capacity of antioxidants.
This kit adopts spectrophotometry, featuring high detection sensitivity, wide linear range and low sample consumption. When the sample volume is 50.0 μL, the spectrophotometric detection shows a good linear relationship for the scavenging rate within the concentration range of 0.0063-0.1 mg/mL.
Component No. | Component Name | Appearance | 48T | Storage | Quantity Per Test |
D1529083A | Extraction Buffer | Liquid | 100.0 mL | 2-8 ℃. | As needed |
D1529083B | Reagent 1 | Powder | 19.2 mg | 2-8 ℃.Store in the dark. | As needed |
D1529083C | Reagent 2 | Powder | 3.31 mg | 2-8 ℃.Store in the dark. | As needed |
D1529083C | Vitamin C (Positive Control) | Powder | 10.0 mg | 2-8 ℃.Store in the dark. | As needed |
Materials to be supplied by user
Visible spectrophotometer (capable of measuring absorbance at 734 nm), 1 mL cuvettes, adjustable‑volume pipettes and pipette tips, low‑temperature centrifuge, ice‑making machine, deionized water, absolute ethanol (optional), homogenizer (for tissue samples).
Operating Procedure
1. Reagent Preparation
1) Reagent 1: Add 5.0 mL of distilled water before use. Dissolve completely to prepare Working Solution 1 for later use.
2) Reagent 2: Add 5.0 mL of distilled water before use. Dissolve completely to prepare Working Solution 2 for later use.
3) Working solution preparation: Mix the pre‑dissolved Working Solution 1 and Working Solution 2 at a 1:1 ratio immediately before use. Keep the mixture at room temperature in the dark for 12–18 h prior to use (the reagent can be stored for three days at 4 °C protected from light). Dilute with extraction solution, a 25‑fold dilution is recommended (adjust according to the detection instrument), so that the absorbance of the blank tube reaches approximately 0.8‑1.1.
Note: Prepare working solution fresh whenever possible. Calculate the required volume of working solution based on the number of test samples.
2. Sample Preparation
1) Plant sample preparation: Weigh approximately 0.1 g tissue, add 1 mL Extraction Buffer, homogenize on ice. Centrifuge at 10,000 g, 4 °C for 10 min. Collect the supernatant and keep on ice for detection.
2) Liquid samples such as red wine and fruit juice: Pipette 100 μL sample solution into 900 μL Extraction Buffer, vortex thoroughly. Centrifuge at 10,000 g, 4 °C for 10 min. Collect the supernatant and keep on ice for detection.
Note: Fresh samples are recommended. If not tested immediately, samples can be stored at -80 °C for one month.
3. Vitamin C (Positive Control) (Optional)
1) Vitamin C (Positive Control): Prepare before use. Dissolve fully in 1 mL deionized water to prepare 10 mg/mL vitamin C solution. Store at 4 °C protected from light for up to two weeks for positive control.
2) Positive control preparation: If linearity verification is required, dilute the 10 mg/mL vitamin C solution 10-fold with deionized water to obtain 1 mg/mL stock, then prepare serial vitamin C solutions of 0.1, 0.05, 0.025, 0.0125, 0.0063, 0 mg/mL for use. Refer to the table below for dilution:
No. | Vitamin C solution & Volume (µL) | Volume of deionized water (µL) | Vitamin C concentration after dilution (mg/mL) |
1 | 1.0 mg/mL: 50 µL | 450 | 0.1 |
2 | 0.1 mg/mL: 200 µL | 200 | 0.05 |
3 | 0.05 mg/mL: 200 µL | 200 | 0.025 |
4 | 0.025 mg/mL: 200 µL | 200 | 0.0125 |
5 | 0.0125 mg/mL: 200 µL | 200 | 0.0063 |
6 | - | 200 | 0 |
4. Sample Assay
1) Power on the spectrophotometer and pre‑warm it for more than 30 min, then set the wavelength to 734 nm. .
2) Sample loading: Set blank wells, sample test wells and positive control wells (standard wells) according to the table below. Add solutions sequentially in order:
Reagent (μL) | Test Well | Blank Well | Positive Control Well (Standard Well) |
Sample supernatant | 50 | - | - |
Vitamin C solution at different concentrations | - | - | 50 |
Deionized water | - | 50 | - |
Working solution | 900 | 900 | 900 |
Mix thoroughly, incubate at room temperature protected from light for 5 min, then measure absorbance at 734 nm. Record values as Atest, Ablank and Astandard, respectively. |
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Note: Positive control wells and blank wells only need to be measured 1-2 times. The capacity of different samples to scavenge ABTS radicals varies greatly. It is recommended to conduct a preliminary experiment with 2-3 samples with anticipated large differences before formal testing. If the scavenging rate is less than 5%, appropriately increase sample loading volume (e.g., increase the volume of liquid sample). If the scavenging rate exceeds 90%, dilute the sample further with Extraction Buffer. Multiply the final result by the dilution factor, or reduce the amount of starting sample for extraction.
5. Calculation of Results
1) Formula for radical scavenging rate of positive control: ABTS radical scavenging rate (%) = (Abₗₐₙₖ -Aₛₜₐₙdₐᵣd)/ Abₗₐₙₖ × 100%
2) Formula for radical scavenging rate of samples: ABTS radical scavenging rate (%) =(Abₗₐₙₖ -Aₜₑₛₜ)/ Abₗₐₙₖ ×100%
Precautions
1) It is recommended to perform a preliminary experiment using 2-3 samples with anticipated large differences before formal measurement.
2) Experimental materials should be as fresh as possible; perform extraction and detection on the same day.
3) Avoid samples that turn blue or near-blue under alkaline conditions, as they will interfere with assay results.
4) Do not add detergents such as Tween, Triton and NP-40, or reducing agents affecting redox reactions including DTT and β-mercaptoethanol to samples.
5) The ABTS radical scavenging capacity varies widely among different samples. To compare scavenging capacity across samples, test equal amounts of samples from the same batch.
6) Samples are recommended to be extracted and assayed on the same day.
7) For personal safety, wear a lab coat and disposable gloves during operation.
8) Use reagents promptly after opening or reconstitution to prevent compromised assay performance.
Appendix 1
The reference standard curve of positive control is shown below. Operate at room temperature following the manual. Measure absorbance of serial VC standard working solutions (0.0063-0.1 mg/mL). Take VC standard working solution concentration as abscissa X and Radical scavenging rate (%) as ordinate Y to establish the standard curve. The values and curve are for reference only.
