Hydroxyl Free Radical Scavenging Capacity Assay Kit (Fenton, Micro Method) - BioReagent, high purity

Cat. No.: P1501782

GRADE & PURITY BioReagent

Overview

The hydroxyl radical (·OH) is the neutral form of the hydroxide ion (OH⁻) and possesses strong oxidizing capacity. Hydroxyl radicals act on biological molecules such as proteins, nucleic acids, and lipids within the body, damaging cellular structure and function, which can lead to metabolic disorders and disease. The hydroxyl radical scavenging capacity is a key indicator of antioxidant ability and is widely used in research on antioxidant health products and pharmaceuticals.

Detection Principle: H₂O₂/Fe²⁺ generates hydroxyl radicals via the Fenton reaction. Salicylic acid effectively captures these generated hydroxyl radicals and reacts with them to produce a purple compound, 2,3-dihydroxybenzoic acid. When a substance capable of scavenging hydroxyl radicals is added, it inhibits the formation of this purple product. Therefore, a darker color indicates a higher hydroxyl radical content, and vice versa. The change in absorbance at 520 nm is measured to calculate the sample's hydroxyl radical scavenging capacity.

Applicable Samples: Animal and plant tissues, serum (plasma), cells, bacteria, cell culture supernatants, fruit juice, honey, urine, and other samples.

P1501782	Component	48 T	96 T	Storage
P1501782A	Ferrous Salt	10 mL	20 mL	2-8℃. Store in the dark.
P1501782B	H₂O₂	5 mL	10 mL	2-8℃. Store in the dark.
P1501782C	Salicylic Acid	10 mL	20 mL	2-8℃. Store in the dark.

Please check the quantities of all components before starting the experiment.

An additional 10% of each component is provided beyond the specified volumes for standard curve preparation or preliminary experiments.

User-Prepared Instruments and Reagents

Type	Name	Notes
Instrument	Microplate Reader	Capable of measuring absorbance at 520 nm.
Consumables	96-well Microplate	Standard microplate.
Reagents	PBS (pH7.4)	For washing samples.
Others	Homogenizer (for tissue samples), Incubator, Ice Box, Refrigerated Centrifuge, Adjustable Micropipettes and Tips	Using a multi-channel pipette can improve efficiency for large-scale assays.

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Notes
Ferrous Salt	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C protected from light. Corrosive. Use appropriate personal protective equipment.
H₂O₂	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C protected from light.
Salicylic Acid	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C protected from light. Irritating to skin and mucous membranes. Use appropriate personal protective equipment.

2. Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month. To compare the hydroxyl radical scavenging capacity of different samples, the dilution factor must be the same for the same batch of samples, and extracts or drugs should be prepared at the same concentration.

2.1 Animal Tissue Samples

Weigh approximately 0.1 g of tissue, add 1 mL of deionized water, 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 Plant Tissue Samples

Weigh approximately 0.1 g of tissue, add 1 mL of deionized water and grind. Sonicate in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.

2.3 Cells or Bacteria

Collect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with pre-cooled PBS, centrifuge, and discard the supernatant. Add 1 mL of deionized water. Sonicate in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.

2.4 Serum (Plasma) and Other Protein-Rich or Turbid Liquids

Take 0.1 mL of sample, add 1 mL of deionized water and mix well. Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.

2.5 Honey, Urine, and Other Clear Liquids with Low Protein Content

Assay directly.

2.6 Extracts or Drugs

Can be prepared to a specific concentration, e.g., 0.5 mg/mL.

3. Assay Steps

3.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 520 nm.

3.2 Assay System Setup: Perform the following operations in a 96-well plate. The Blank and Standard wells only need to be set up 1-2 times. Each test well requires a corresponding control well.

Reagent	Blank Well (μL)	Standard Well (μL)	Test Well (μL)	Control Well (μL)
Ferrous Salt	40	40	40	40
H₂O₂	0	40	40	0
Deionized Water	120	80	40	80
Salicylic Acid	40	40	40	40
Sample	0	0	40	40

3.3 Absorbance Measurement: Mix well, incubate at 37°C for 20 minutes. Read the absorbance at 520 nm, recorded as A _blank , A _standard , A _test , and A _control respectively.

4. Calculation of Results

Both the derived formula and the simplified formula provided below are equivalent.

4.1 Data Processing

Calculate ΔA _test = A _test - A _control

Calculate ΔA _standard = A _standard - A _blank

4.2 Calculation of Hydroxyl Radical Scavenging Rate

Hydroxyl Radical Scavenging Rate D% = (ΔA _standard - ΔA _test ) / ΔA _standard × 100%

5. Representative Results

Example: 0.1 g of nectarine pulp was taken and assayed according to the procedure using a 96-well plate.

Measured: ΔA _standard = A _standard - A _blank = 1.020 - 0.051 = 0.969

ΔA _test = A _test - A _control = 0.465 - 0.052 = 0.413

Calculated Hydroxyl Radical Scavenging Rate D% = (0.969 - 0.413) / 0.969 × 100% = 57.38%

Precautions

1. Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.

2. For tissue samples, cell samples, etc., results can be normalized between samples by measuring protein concentration. Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.

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

4. Biochemical reagents are generally irritating and potentially biologically toxic. For your safety and health, implement appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, and head covers. Perform experiments in a fume hood or biosafety cabinet.

5. This product is for research use only. Not for use in clinical diagnosis.

Frequently Asked Questions

Q: What should I do if the measured ΔA _test for the sample is too high or too low?

A: If ΔA _test < 0.02, appropriately increase the sample volume and re-run the assay. If ΔA _test > ΔA _standard , further dilute the sample with deionized water or reduce the amount of sample used for extraction, and re-run the assay.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.