Catalase(CAT) Activity Assay Kit(UVspectrophotometry)(UVspectrophotometry)
enzyme labeling method
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The Catalase (CAT) Activity Assay Kit is widely used in research and diagnostics to measure catalase enzyme activity in biological samples. It is particularly valuable in studies related to oxidative stress, as catalase plays a critical role in breaking down hydrogen peroxide into water and oxygen, thus protecting cells from oxidative damage. This kit is commonly applied in fields such as biochemistry, molecular biology, and toxicology to assess the antioxidant capacity of tissues, cells, or enzymes. It is also utilized in agricultural research to evaluate plant stress responses and in pharmaceutical studies to test the efficacy of antioxidant drugs. The UV spectrophotometry method, which monitors the decrease in hydrogen peroxide absorbance at 240 nm, ensures accurate and reliable quantification of catalase activity, making it a preferred choice for high-throughput screening and experimental analysis.
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| Test Parameter | Specification |
|---|---|
| Performance Test | PASS |
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Catalase (CAT) Activity Assay Kit - Microplate Method (Ultraviolet Absorption Method)
Product code: 112869
Product Introduction
Catalase (CAT, EC 1.11.1.6) is widely present in animals, plants, microorganisms, and cultured cells. It is an important H2O2-scavenging enzyme and plays a key role in the reactive oxygen species scavenging system.
H2O2has a characteristic absorption peak at 240 nm. CAT decomposes H2O2, causing the absorbance of the reaction solution at 240 nm to decrease over time. CAT activity is calculated from the rate of absorbance change.
Reference image note: The sample shown was 500-fold diluted pig liver. Actual readings may vary depending on test conditions and the instrument used. Data shown in the source figure are for reference only.
Product Packing List
| Size | Code | Component | Quantity |
|---|---|---|---|
| 100T | 112869.1 | Reagent I | 24 mL |
| 100T | 112869.2 | Reagent II | 55 μL × 2 |
| 100T | 112869.3 | Extraction Solution | 110 mL |
| 100T | 112869.m | Instruction Manual | 1 copy |
Quality Standards and Safety Information
| Raw Material or Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent I | -- | -- |
| Reagent II | -- | -- |
| Extraction Solution | -- | -- |
Transportation and Storage
| Transportation | Transport with ice packs. |
|---|---|
| Storage | Store at 2-8℃. Shelf life is 180 days. |
Instructions for Use
1. Sample Preparation
1.1 Bacteria or Cells
- Collect bacteria or cells into a centrifuge tube.
- Centrifuge and discard the supernatant.
- Add 400 μL extraction solution for every 2,000,000 bacteria or cells.
- Disrupt the bacteria or cells by ultrasonication at 20% power: ultrasonication for 3 s, interval for 10 s, repeat 30 times.
- Centrifuge at 8000 g and 4℃ for 10 min.
- Collect the supernatant and keep it on ice for testing.
1.2 Tissue
- Weigh approximately 0.1 g tissue.
- Add 1 mL extraction solution.
- Homogenize in an ice bath.
- Centrifuge at 8000 g and 4℃ for 10 min.
- Collect the supernatant and keep it on ice for testing.
1.3 Serum or Plasma
Test serum or plasma samples directly.
2. Assay Procedure
- Before use, add 11 mL Reagent I to each vial of Reagent II and mix thoroughly to prepare the working solution.
- Before measurement, place the CAT assay working solution in a water bath at 37℃ for mammals or 25℃ for other species for 10 min.
- Add 200 μL CAT assay working solution to a 96-well UV plate.
- Add 10 μL crude enzyme solution and mix thoroughly.
- Immediately measure the initial absorbance at 240 nm at room temperature as A1.
- Measure the absorbance after 1 min as A2.
- Calculate ΔA = A1- A2.
CAT Activity Calculation
1. Serum or Plasma
Unit definition: The amount of enzyme that catalyzes the degradation of 1 μmol H2O2per minute in each mL of serum or plasma in the reaction system is defined as one enzyme activity unit.
CAT (U/mL) = [ΔA × Vtotal reaction÷ (ε × d) × 106] ÷ Vsample÷ T = 802.751 × ΔA
2. Tissue
2.1 Calculated by Sample Protein Concentration
Unit definition: The amount of enzyme that catalyzes the degradation of 1 μmol H2O2per minute in each mg of tissue protein in the reaction system is defined as one enzyme activity unit.
CAT (U/mg prot) = [ΔA × Vtotal reaction÷ (ε × d) × 106] ÷ (Vsample× Cpr) ÷ T = 802.751 × ΔA ÷ Cpr
2.2 Calculated by Sample Fresh Weight
Unit definition: The amount of enzyme that catalyzes the degradation of 1 μmol H2O2per minute in each g of tissue in the reaction system is defined as one enzyme activity unit.
CAT (U/g fresh weight) = [ΔA × Vtotal reaction÷ (ε × d) × 106] ÷ (W × Vsample÷ Vtotal sample) ÷ T = 802.751 × ΔA ÷ W
3. Bacteria or Cells
Unit definition: The amount of enzyme that catalyzes the degradation of 1 μmol H2O2per minute in each 10,000 bacteria or cells in the reaction system is defined as one enzyme activity unit.
CAT (U/104cells) = [ΔA × Vtotal reaction÷ (ε × d) × 106] ÷ (500 × Vsample÷ Vtotal sample) ÷ T = 1.6055 × ΔA
Formula Parameters
| Vtotal reaction | Total volume of the reaction system, 0.21 × 10-3L |
|---|---|
| ε | H2O2molar extinction coefficient, 43.6 L/mol/cm |
| d | 1 mL optical path length of the quartz cuvette, 0.6 cm |
| Vsample | Volume of sample added, 0.01 mL |
| Vtotal sample | Volume of extraction solution added, 1 mL |
| T | Reaction time, 1 min |
| W | Sample mass, g |
| Cpr | Supernatant protein concentration, mg/mL |
| 500 | Total number of cells or bacteria, 5,000,000 |
| 106 | Unit conversion factor, 1 mol = 106μmol |
Precautions
- Before formal measurement, select 2-3 samples with large expected differences for preliminary testing. This 100T kit can test 96 samples.
- Required instruments and supplies are not provided: benchtop centrifuge, microplate reader, 96-well UV plate, adjustable pipette, mortar or homogenizer, ice, and distilled water.
- If a large number of bubbles form in the reaction solution, the enzyme activity of the sample is too high. Dilute the sample with distilled water before measurement.
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