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.

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Instructions for Use

1. Sample Preparation

1.1 Bacteria or Cells

1. Collect bacteria or cells into a centrifuge tube.
2. Centrifuge and discard the supernatant.
3. Add 400 μL extraction solution for every 2,000,000 bacteria or cells.
4. Disrupt the bacteria or cells by ultrasonication at 20% power: ultrasonication for 3 s, interval for 10 s, repeat 30 times.
5. Centrifuge at 8000 g and 4℃ for 10 min.
6. Collect the supernatant and keep it on ice for testing.

1.2 Tissue

1. Weigh approximately 0.1 g tissue.
2. Add 1 mL extraction solution.
3. Homogenize in an ice bath.
4. Centrifuge at 8000 g and 4℃ for 10 min.
5. Collect the supernatant and keep it on ice for testing.

1.3 Serum or Plasma

Test serum or plasma samples directly.

2. Assay Procedure

1. Before use, add 11 mL Reagent I to each vial of Reagent II and mix thoroughly to prepare the working solution.
2. Before measurement, place the CAT assay working solution in a water bath at 37℃ for mammals or 25℃ for other species for 10 min.
3. Add 200 μL CAT assay working solution to a 96-well UV plate.
4. Add 10 μL crude enzyme solution and mix thoroughly.
5. Immediately measure the initial absorbance at 240 nm at room temperature as A₁.
6. Measure the absorbance after 1 min as A₂.
7. 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 × V_{total reaction}÷ (ε × d) × 10⁶] ÷ V_sample÷ 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 × V_{total reaction}÷ (ε × d) × 10⁶] ÷ (V_sample× 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 × V_{total reaction}÷ (ε × d) × 10⁶] ÷ (W × V_sample÷ V_{total 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 × V_{total reaction}÷ (ε × d) × 10⁶] ÷ (500 × V_sample÷ V_{total sample}) ÷ T = 1.6055 × ΔA

Formula Parameters

Precautions

1. Before formal measurement, select 2-3 samples with large expected differences for preliminary testing. This 100T kit can test 96 samples.
2. Required instruments and supplies are not provided: benchtop centrifuge, microplate reader, 96-well UV plate, adjustable pipette, mortar or homogenizer, ice, and distilled water.
3. 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.

Visual Reference