Catalase (CAT) Activity Assay Kit (MAES0343)

Materials Prepared by Users

Instruments: Spectrophotometer (405 nm), micropipettor, vortex mixer, incubator

Reagents: Double distilled water, normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4)

1. Incubate buffer solution and substrate at 37°C for 10 min before use.
2. Preparation of chromogenic application solution: Dissolve one vial of chromogenic agent with 60 mL of double distilled water. (If there is sediment at the bottom, directly use the supernatant for testing, as this will not affect the result). Store at 2-8°C for 3 months.
3. Clarificant will freeze when cold; please warm it in a 37°C water bath until clear.

Serum and Plasma: Detect directly. If not analyzed on the same day, serum or plasma can be stored at -80°C for one month.

Tissue Sample:

1. Harvest the amount of tissue needed for each assay (initial recommendation: 20 mg).
2. Wash tissue in cold PBS (0.01 M, pH 7.4).
3. Homogenize 20 mg tissue in 180 μL PBS (0.01 M, pH 7.4) with a dounce homogenizer at 4°C.
4. Centrifuge at 10,000×g for 10 min at 4°C to remove insoluble material. Collect supernatant and keep on ice for detection.
5. Meanwhile, determine the protein concentration of supernatant (MAES0177 is recommended for animal tissue samples; MAES0126 is recommended for plant tissue samples).

Cells:

1. Harvest the number of cells needed for each assay (initial recommendation: 1×10⁶ cells).
2. Wash cells with PBS (0.01 M, pH 7.4).
3. Homogenize 1×10⁶ cells in 200 μL PBS (0.01 M, pH 7.4) with an ultrasonic cell disruptor at 4°C.
4. Centrifuge at 10,000×g for 10 min at 4°C to remove insoluble material. Collect supernatant and keep on ice for detection.
5. Meanwhile, determine the protein concentration of supernatant (MAES0177).

1. Test tubes can be prepared and labeled in advance. After incubating at 37°C for 10 min, add samples and buffer solution, then incubate the test tube at 37°C for 5 min.
2. Dilute samples to the optimal concentration for detection if the CAT activity of samples exceeds the detection range.
3. The reaction time must be accurate when substrate is added.

Note: For serum or plasma samples, a* is 0.1 mL. For cell homogenate and tissue homogenate, a* is 0.05 mL.

1. Control tube: Add 1 mL of buffer solution into 5 mL EP tubes. Sample tube: Add a* mL of sample and 1 mL of buffer solution into 5 mL EP tubes.
2. Incubate at 37°C for 5 min.
3. Add 0.1 mL of substrate into each tube, mix well and react at 37°C for exactly 1 min.
4. Control tube: Add 1 mL of chromogenic application solution, 0.1 mL of clarificant and a* mL of sample, mix well. Sample tube: Add 1 mL of chromogenic application solution and 0.1 mL of clarificant, mix well.
5. Stand for 10 min at room temperature. Set to zero with double distilled water and measure the OD values of each tube at 405 nm with 0.5 cm optical path cuvette.

Intra-assay Precision

Inter-assay Precision

Recovery

Sensitivity: The analytical sensitivity of the assay is 0.27 U/mL CAT. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.