Plant peroxidase, a member of the superfamily of peroxidase, catalyzes the redox reaction between H2O2 and various reductants. The plant peroxidase has the same general structure and consists of iron porphyrin IX and ten α-helixes. Based on the difference of primary structure, the superfamily of plant peroxidase can be divided into three types: class I (intracellular type), class II (extracellular type of fungi) and class III (secreted type of plant).

1. The reaction time must be controlled strictly.

2. The light should be prevented during the experiment, so as to avoid the phenomenon that the difference between the multiple wells is too large.

3. Don’t take the precipitate when take the supernatant for measuring the OD value to avoid the effect of precipitate to OD value.

4. The step of measuring the OD value must be finished in 30 min.

5. If the OD value of sample tube is more than 0.6, the sample must be diluted and test again.

6. During the detection, the cuvettes should be washed, so as to avoid the residual water in the cuvette to affect the results.

1.     Sample tube: Add 2.4 mL of Reagent 1, 0.3 mL of Chromogenic agent, 0.2 mL of reagent 3 application solution and 0.1 mL of Sample into a 5 mL EP tube.

Control tube: Add 2.4 mL of Reagent 1, 0.3 mL of Chromogenic agent, 0.2 mL of Double-distilled water and 0.1 mL of Sample into a 5 mL EP tube.

2.     Oscillate fully with the vortex mixer, then incubate the tubes at 37℃ for 30 min accurately.

3.     Add 1.0 mL of Reagent 4 application solution into each tube, mix fully and centrifuge at 2300 g for 10 min. Take the supernatant for detect the OD value.

4.     Set the spectrophotometer to zero with double-distilled water and measure the OD values of each tube at 420 nm with 1 cm optical path cuvette (This step must be finished in 30 min).