Catalase is a key antioxidant enzyme with iron porphyrin as its prosthetic group, widely present in animals, plants, microorganisms, and cultured cells. Its main function is to catalyze the decomposition of hydrogen peroxide into water and oxygen. H₂O₂ is a reactive oxygen species produced by cellular aerobic metabolism, which can directly or indirectly oxidize nucleic acids, proteins, lipids, and other biological macromolecules, leading to cell damage, accelerated aging, and disintegration. Catalase protects cells from the toxicity of H₂O₂ by scavenging it, and is one of the key enzymes in the biological defense system.
The principle of this kit is as follows: Catalase catalyzes the decomposition of the substrate hydrogen peroxide (H₂O₂) into water and oxygen, consuming a defined amount of H₂O₂ in the system. The remaining H₂O₂ reacts with Amplex Red in the presence of HRP to generate the red fluorescent compound resorufin, and the fluorescence intensity is proportional to the remaining H₂O₂ concentration. Therefore, higher enzyme activity results in more substrate consumption and weaker fluorescence signal. Subtract the sample fluorescence value from that of the control well without enzyme; the resulting difference (i.e., the fluorescence decrease value) directly reflects the amount of H₂O₂ decomposed, and this decrease is proportional to the enzyme activity. A standard curve is established based on the linear relationship between the fluorescence decrease and the activity of the standard, thereby allowing quantification of enzyme activity in the sample.
Components
A1522296 | Components | Appearance | 100 T | Storage | Quantity Per Test |
A1522296A | Lysis Buffer | Liquid | 30 mL | -20°C | As needed |
A1522296B | Assay Buffer | Liquid | 30 mL | -20°C | As needed |
A1522296C | Amplex Red | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
A1522296D | Horseradish Peroxidase | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
A1522296E | Catalase standard (200 U/mL) | Liquid | 200 μL | -20°C. Store in the dark | 2 μL |
A1522296F | Hydrogen peroxide (1 M) | Liquid | 1 mL | -20°C. Store in the dark | 2 μL |
Usage Protocol
1. Sample Preparation
1) Blood samples. For properly prepared serum or plasma samples, they can be directly assayed or diluted with assay buffer before measurement.
2) Cell samples. Collect cells, wash once with PBS. Add lysis buffer at a ratio of 100–200 μL per 1 × 10⁶ cells, and lyse on ice for 5–10 minutes or by sonication homogenization. Centrifuge at approximately 12,000 × g for 5 minutes at 4 °C, and collect the supernatant for subsequent analysis.
3) Tissue samples. Add lysis buffer at a ratio of 100 μL per 10 mg of tissue, and homogenize under low‑temperature conditions such as on ice. Centrifuge at approximately 12,000 × g for 3–5 minutes at 4 °C, and collect the supernatant for subsequent analysis.
All of the above operations should be performed at 4 °C or on ice. If the prepared cell or tissue samples cannot be analyzed immediately, they may be stored at –20 °C or –80 °C.
2. Preparation of Catalase Standard Curve
Take six EP tubes. Add 2 µL of catalase standard (200 U/mL) and 398 µL of assay buffer or lysis buffer (use assay buffer for samples that do not require lysis treatment; use lysis buffer for samples that require lysis treatment) to the first EP tube to dilute the catalase to 1000 mU/mL. Add 200 μL of assay buffer or lysis buffer to each of the remaining tubes. Transfer 200 μL from the first tube to the second tube, and continue this serial dilution through the subsequent tubes. The last tube contains 200 μL of assay buffer or lysis buffer. The resulting catalase concentrations in each tube are 1000, 500, 250, 125, 62.5, and 0 mU/mL, respectively.
3. Preparation of Hydrogen Peroxide Working Solution
Since hydrogen peroxide is not very stable, its actual concentration must be determined by the user before use for calibration. Dilute the approximately 1 M hydrogen peroxide solution 100‑fold with water to obtain a concentration of approximately 10 mM, and measure A₂₄₀. The A₂₄₀ measurement can be performed by any of the following methods: ordinary UV spectrophotometer, micro‑volume UV spectrophotometer, or 96‑well UV microplate reader. Among these, the hydrogen peroxide concentration measured using a cuvette is closest to the actual concentration. Taking calibration using a cuvette with a 1 cm pathlength as an example, calculate the hydrogen peroxide concentration using the following formula: Hydrogen peroxide concentration (mM) = 22.94 × A₂₄₀. Multiply the result by the dilution factor of 100 to obtain the calibrated concentration of hydrogen peroxide.
Based on the calibrated hydrogen peroxide concentration, dilute the hydrogen peroxide to prepare a 200 μM hydrogen peroxide working solution. For sample detection, add 25 μL of the hydrogen peroxide working solution to each standard well and sample well. Prepare the amount needed, and use it immediately after preparation; it should be used as soon as possible.
4. Preparation of Chromogenic Solution
Prepare an appropriate amount of Amplex Red Reaction Working Solution based on a volume of 50 μL per reaction. Mix 46 μL of Assay Buffer, 2 μL of Amplex Red, and 2 μL of Horseradish Peroxidase to prepare 50 μL of Amplex Red Reaction Working Solution. Prepare an appropriate amount according to the number of samples to be tested (including standards). It is recommended to prepare the working solution freshly before use. Refer to the table below for specific preparation methods:
Number of samples | 1 | 10 | 20 | 50 |
Assay Buffer | 46 µL | 460 µL | 920 µL | 2300 µL |
Amplex Red | 2 µL | 20 µL | 40 µL | 100 µL |
Horseradish Peroxidase | 2 µL | 20 µL | 40 µL | 100 µL |
Total volume | 50 µL | 500 µL | 1000 µL | 2500 µL |
5. Sample Determination
1) Pipette 25 μL of standards and samples into a black 96-well plate; wells containing only lysis buffer or assay buffer shall be set as blank controls. Subsequently, add 25 μL of hydrogen peroxide substrate working solution to standard wells and sample wells. Background control wells may be prepared by replacing 25 μL hydrogen peroxide substrate working solution with 25 μL assay buffer. Incubate the plate for 20 min at room temperature in the dark.
2) Add 50 µL chromogenic substrate to standard wells and sample wells, mix thoroughly, and incubate for another 20 min at room temperature with light shielding.
3) Perform fluorescence measurement with excitation wavelength set at 530 nm and emission wavelength at 590 nm to record fluorescence intensity.
6. Result Calculation
The fluorescence value of the blank control well (without catalase) in the standard curve is A1, and the fluorescence value of the standard wells is A2; then the fluorescence decrease value for each standard well is A1 – A2. Plot a standard curve with catalase activity as the abscissa and the fluorescence decrease value as the ordinate.
The fluorescence decrease value of the sample is obtained by subtracting the sample fluorescence value from the fluorescence value of the blank control well (without catalase) in the standard curve. Substitute the fluorescence decrease value of the sample well into the standard curve to obtain the catalase activity in the sample.
7. Results Presentation
Standard curve
Precautions
1. Amplex Red is relatively unstable in the air. Use it as soon as possible after opening, and take care to protect it from light during use.
2. Amplex Red must be completely thawed and equilibrated to room temperature before use; otherwise, the detection results may be affected.
3. To reduce errors caused by fluorescence background from the diluent, the diluent for samples and standards should be selected according to the sample type. When the sample is a cell or tissue lysate prepared with lysis buffer, lysis buffer should be used for dilution; for other samples such as blood, assay buffer should be used for dilution.
4. For fluorescence detection, a black 96‑well plate is recommended.
5. For your safety and health, please wear a lab coat and disposable gloves during operation.
