Hydrogen Peroxide (H₂O₂) Content Assay Kit (TTS, Micro Method)

Cat. No.: H1505503

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Application

Cell Metabolism

Overview

Hydrogen peroxide (H₂O₂) is the most common reactive oxygen species (ROS) molecule in living organisms. It is a byproduct of active oxygen metabolism, primarily generated by enzymes like SOD and XOD, and degraded by enzymes such as CAT and POD. H₂O₂ is not only a significant ROS but also a hub for the interconversion of various reactive oxygen species. On one hand, H₂O₂ can directly or indirectly oxidize biological macromolecules like nucleic acids and proteins within cells, damaging cell membranes and thereby accelerating cellular aging and disintegration. On the other hand, H₂O₂ is a key regulatory factor in many oxidative stress responses. It can activate factors like NF-κB, and these H₂O₂-related signaling pathways are associated with numerous diseases such as asthma, inflammatory arthritis, arteriosclerosis, and neurodegenerative disorders. H₂O₂ is also closely linked to processes like cell apoptosis and proliferation.

Assay Principle

H₂O₂ reacts with titanium sulfate to form a yellow peroxide-titanium complex precipitate. This precipitate dissolves in strong acid, and the intensity of the yellow color is linearly proportional to the H₂O₂ concentration within a certain range. The absorbance at 412 nm is measured using a microplate reader. This kit is primarily used to detect H₂O₂ content in samples such as plant tissues, serum, and plasma. It is intended for research use only and not suitable for clinical diagnosis or other purposes.

H1505503	Component	100T	Storage
H1505503A	H₂O₂ Stock Solution	1 mL	2-8℃. Store in the dark.
H1505503B	Alkaline Matrix Solution	10.5 mL	RT.
H1505503C	Titanium Sulfate	0.3 g	RT.
H1505503D	Acidic Matrix Solution	100 mL	RT.

Required Materials Not Provided

1. Distilled water, Acetone

2. Homogenizer or mortar and pestle, Refrigerated centrifuge, Microplate reader, 96-well plate

Experimental Procedure

1. Sample Preparation

1.1 Plant Samples

• Take fresh plant tissue (under normal or stress conditions), wash clean, dry, and mince.

• Quickly weigh 5 g, add 5 mL of pre-chilled acetone.

• Homogenize or grind rapidly in an ice bath.

• Centrifuge at 12,000 g for 20 min at 4°C.

• Collect the supernatant, measure the total volume of the extract, and store at 4°C for later use.

1.2 Plasma, Serum, and Urine Samples

• Plasma and serum, prepared by conventional methods, can be directly used for assay with this kit. Store at 4°C for H₂O₂ determination.

1.3 High-Activity Samples

• If the sample contains a high concentration of H₂O₂, it can be appropriately diluted with acetone.

2. Preparation of 10 mM H₂O₂ Standard Solution

• Since H₂O₂ is not very stable, its actual concentration needs to be determined before use.

• The H₂O₂ Stock Solution provided has an H₂O₂ concentration of approximately 1 M. Dilute it 100-fold with distilled water to achieve ~10 mM.

• Zero the spectrophotometer with distilled water and measure the absorbance at 240 nm (A₂₄₀).

• Calculate the actual H₂O₂ concentration using the formula: H₂O₂ concentration (mM) = A₂₄₀ × 22.94.

• Then, dilute the H₂O₂ Stock Solution with acetone to prepare a 10 mM H₂O₂-acetone standard solution.

• Prepare serial dilutions according to the table below (common range 0.3-3 mM, i.e., Tubes 1-5):

Tube	10 mM H₂O₂-Acetone Standard (mL)	Pre-chilled Acetone (mL)	Final H₂O₂ Concentration (mM)
1	0.03	0.97	0.3
2	0.05	0.95	0.5
3	0.08	0.92	0.8
4	0.1	0.9	1
5	0.3	0.7	3
6	0.5	0.5	5
7	0.8	0.2	8

3. Preparation of Titanium Sulfate Solution

• Add 0.3 g of Titanium Sulfate to 6 mL of distilled water to make a 5% Titanium Sulfate solution. Store at 4°C.

4. H₂O₂ Assay Setup

• Set up Blank, Standard, and Test tubes as per the table below.

• Add reagents in the specified order, avoiding bubbles.

• If the sample H₂O₂ concentration is too high, reduce the sample volume or dilute appropriately before assay. Duplicate tubes are recommended.

Reagent (mL)	Blank Tube	Standard Tube	Test Tube
Pre-chilled Acetone	0.5	—	—
Serial H₂O₂-Acetone Standards	—	0.5	—
Sample Extract	—	—	0.5
Alkaline Matrix Solution	0.1	0.1	0.1
Titanium Sulfate Solution	0.05	0.05	0.05

Note: When adding Alkaline Matrix Solution and Titanium Sulfate Solution, add them directly into the liquid, avoiding the tube walls.

5. H₂O₂ Measurement

• Mix well and let stand at room temperature for 5 minutes.

• Centrifuge at 12,000 g for 15 minutes.

• Discard the supernatant carefully, retaining the precipitate.

• If necessary, wash the precipitate repeatedly with pre-chilled acetone.

• Add 1 mL of Acidic Matrix Solution to the precipitate in each tube.

• Shake to dissolve the precipitate completely.

• Transfer 300 µL of each tube's solution to a 96-well plate.

• Zero the microplate reader using the blank solution.

• Measure the absorbance of standard and test wells at 412 nm.

6. Calculation of Results

6.1 Standard Curve Plotting

• Plot the standard curve using the serial H₂O₂-acetone standard concentrations (0.3, 0.5, 0.8, 1, 3, 5, 8 mM) on the X-axis and their corresponding absorbances on the Y-axis. Determine the regression equation.

6.2 Sample H₂O₂ Concentration Calculation

• Substitute the absorbance of the test sample into the regression equation to find its H₂O₂ concentration (C₀).

• For tissue samples: H₂O₂ (mmol/g weight) = (C₀ × V_T × N) / m

• For liquid samples: H₂O₂ (mmol/L) = C₀ × N

Parameter Definitions:

C₀: H₂O₂ concentration (mM) obtained from the standard curve based on the sample's absorbance.

V_T: Total volume of the test sample extract (L).

m: Sample mass (g).

N: Sample dilution factor.

Results Presentation

Standard Curve Example: When measured according to the instructions at room temperature, the absorbance values for the serial standards and a typical standard curve are as follows (for reference only). A standard curve was plotted using H₂O₂ standards (0.1, 0.3, 0.5, 0.8, 1, 3, 5, 8, 10 mM). Note: Standard concentrations that are too high or too low may affect the accuracy of the standard curve.

H₂O₂ Standard (mM)	0.1	0.3	0.5	0.8	1	3	5	8	10
Absorbance	0.009	0.065	0.141	0.216	0.264	0.812	1.262	2.010	2.355

Note: H₂O₂ concentrations below 0.3 mM are basically colorless, 0.3~1 mM appear yellow, and 3~10 mM appear orange-yellow. Refer to the image below for expected results. 

Precautions

1. This kit can also be used with a spectrophotometer, but the number of samples processed will be correspondingly reduced.

2. When adding Alkaline Matrix Solution and Titanium Sulfate Solution, add them directly into the solution, avoiding the tube walls.

3. Dissolving the yellow peroxide-titanium complex precipitate in the Acidic Matrix Solution takes time. Ensure it is completely dissolved, otherwise results may be affected.

4. The H₂O₂ Stock Solution and Alkaline Matrix Solution should be tightly sealed to prevent evaporation, as efficiency may decrease otherwise.

5. The H₂O₂ Stock Solution and Acidic Matrix Solution are corrosive. Handle with care.

6. The Titanium Sulfate solution should be used soon after preparation. If not used immediately, it can be stored short-term at 4°C. Alternatively, weigh an appropriate amount of powder and prepare a 5% solution as needed.

7. For your safety and health, wear lab coats and disposable gloves during operation.

8. Use reagents promptly after opening to avoid affecting subsequent experimental results.

Specifications

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. H1505503A: Store at 2-8℃ long term (12 months). Store in the dark. H1505503B, H1505503C, H1505503D: Store at room temperature long term (12 months).

Storage

Store at 2-8°C, Protected from light, Room temperature

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Chemical and Physical Properties

• Sensitivity: Light-sensitive