Catalase (CAT) Activity Assay Kit (for Biochemical Assay Kit)

Product Information

Product Description/Introduction

Catalase (CAT) is an antioxidant enzyme widely found in plants, animals and microorganisms, which scavenges hydrogen peroxide from the body by catalyzing the decomposition of hydrogen peroxide (H2O2) into water and oxygen, and is one of the key enzymes in the biological defense system.

CAT catalyzes the decomposition reaction of H2O2, which can be quickly terminated by ammonium molybdate, and the interaction of the remaining H2O2 and ammonium molybdate produces a yellowish complex. The complex can be measured at a wavelength of 405 nm, and the activity of CAT can be calculated based on the amount of change in the complex.

Storage and Shipping Conditions

Ship with wet ice; Store at 2-8 away from light for 6 months.

Product Content

Assay Protocol / Procedures

1. Sample Preparation:

1.1. Plasma and serum samples can be directly used for the detection of CAT activity;

1.2. Tissue samples: Use suitable buffer (PBS, saline, etc.) for homogenization and lysis, where the ratio of tissue to reagent is 1 to 9 (0.1 g : 0.9 mL); After homogenization and lysis, centrifuge at 4, 10000 g for 10-15 min, and take the supernatant for the subsequent CAT activity assay;

1.3. Cell samples: After cell collection, cells can be resuspended with a suitable buffer solution, approximately 500 μL of PBS or saline per 1-2 × 106 cells; after homogenization, centrifuge the cells at 10,000 g for 10-15 min at 4°C, and remove the supernatant for subsequent CAT assay.

2. Pre-test preparation:

2.1. Hydrogen peroxide standard preparation: According to the need, the use of ultrapure water to hydrogen peroxide standard (9.9 mol/L) for the gradient dilution (such as dilution to 100/50/25/12.5/6.25/3.125/0 mM), and for subsequent testing of sample under test, for standard curve method of data analysis;

2.2. Color developing working solution preparation: Color developing agent: Clarifying agent=10:1 to prepare the Color developing working solution (ready to use);

Note: Clarifying agent will solidify at low temperature, can be dissolved in water bath at 37; The precipitation of color developing agent during storage is a normal phenomenon, and it does not affect the use effect after being redissolved in water bath at 75.

3. CAT detection:

3.1. Conduct detection according to the following table:

4. Data analysis:

Before data analysis, the protein concentration of the tissue or cell samples to be tested should be calculated. The protein concentration can be determined by BCA Protein Quantification Kit (G2026) （1 mmol/L=1 μmol/mL）.

Definition: One unit of viability is defined as 1 μmol of H2O2 decomposed per minute per milliliter of serum plasma or per milligram of histone at 37°C;

4.1. Plotting standard curves from gradient dilution data: Y=a X+ b;

4.2. CAT activity calculation of serum and plasma samples:

Unit sample CAT viability (U/mL) = （ΔA-b）÷a×V1÷1^*÷V2×f;

4.3. CAT activity calculation of tissue and cell samples:

Unit sample CAT activity (U/mgprot) =（ΔA-b）÷a×V1÷1^*÷V2×f÷

Y is the standard group value - blank group value (0 mM standard group value); X is the hydrogen peroxide concentration of the standard; a is the slope of the standard curve; b is the intercept of the standard curve; ΔA is: OD value of the control group - OD value of the assay group; V1 is the volume of the substrate (0.01 mL); V2 is the volume of the sample (0.01 mL); 1^* is the reaction time (1 min);  is the protein concentration of the sample ( mgprot/mL); f is the dilution factor.

Note

1. Hydrogen peroxide is unstable but does not affect the relative trend of results. If absolute data is required, the customer can calibrate the concentration of hydrogen peroxide standard in this product or provide their own hydrogen peroxide reagent.

2. If there are bubbles in the holes, blow them gently with the pipette to avoid affecting the test results.

3. For data accuracy, ensure that the standard curve R2> 0.99.

4. For your health and safety, please wear lab coat and gloves during operation.

Appendix: Technical parameters

For Research Use Only!

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