1.  Because H2O2 is very unstable, the actual concentration of H2O2 needs to be calibrated before use. This is the key point to the success of the experiment.

2.  Prevent the formulation of bubbles when the supernatant is transferred into the microplate.

                                                                               [Note]: A-H, standard wells; S1-S40, sample wells; S1'-S40', control wells. 

For samples:

1)    Control tube: Add 200 μL of Reagent 1 into the 1.5 mL EP tubes.

Sample tube: Add 20 μL of sample and 200 μL of Reagent 1 into the 1.5 mL EP tubes.

2)    Incubate at 37℃ for 5 min.

3)    Add 20 μL of Reagent 2 into each tube, mix fully and react at 37℃ for 1 min accurately.

4)    Sample tube: Add 200 μL of Reagent 3 application solution and 20 μL of Reagent 4, mix fully.

Control tube: Add 200 μL of Reagent 3 application solution, 20 μL of Reagent 4 and 20 μL of sample, mix fully.

5)    Stand at room temperature for 10 min and take 200 μL of reaction solution to the Microplate with a micropipette.

6)    Measure the OD value at 405 nm with Microplate Reader.

For standard solution:

1)    Add 20 μL of H2O2 standard solution with different concentrations to the 1.5 mL EP tubes respectively.

2)    Sequentially add 200 μL of Reagent 1, 20 μL of double distilled water, 200 μL of Reagent 3 application solution and 20 μL of Reagent 4, mix fully.

3)    Stand at room temperature for 10 min and take 200 μL of reaction solution to the Microplate with a micropipette.

4)    Measure the OD value at 405 nm with Microplate Reader.

For samples:

      For standard solution:

Dilute1 mmol/mL H2O2 standard solution with double distilled water to a serial concentration. The recommended dilution gradient is as follows:60, 50, 40, 30, 20, 10, 0 μmol/L. Then carry the assay according to the operation table.

Plot the standard curve by using OD value of standard and correspondent concentration as y-axis and x-axis respectively. Create the standard curve with graph software (or EXCEL). The concentration of the sample can be calculated according to the formula based on the OD value of sample. The standard curve is: y= ax + b.

Example analysis

For Rat liver tissue, dilute the 10% rat liver tissue homogenate with PBS (0.01 M, pH 7.4) for 160 times, take 0.02 mL diluted sample, carry the assay according to the operation table.

The results are as follows:

standard curve: y =0.0026 x + 0.0022, the average OD value of the sample tube is 0.442, the average OD value of the control tube is 0.612, the concentration of protein in sample is 12.38 mgprot/mL, and the calculation result is:

CAT activity (U/mgprot)=(0.612-0.442)÷0.0026×100÷12.38=528.15U/mgprot

Detect rat serum (dilute for 2 times), 10% rat liver tissue homogenate (the concentration of protein is 12.38 mgprot/mL dilute for 100 times), 10% mouse muscle tissue homogenate (the concentration of protein is 3.10 mgprot/mL) and HepG2 cells (the concentration of protein is 8.06 mgprot/mL, dilute for 10 times) according to the protocol, the result is as follows: