The kit can be used to measure the APX activity in plant tissue samples.

Detection significance

Ascorbate Peroxidase (APX) is composed of three subunits of cytoplasm, peroxisome and chloroplast isozymes. APX is unique to plants and algae, and is necessary to protect chloroplasts and other cellular components from damage by hydrogen peroxide and hydroxyl radicals. APX is the core component of ascorbic acid-glutathione cycle, which is the main hydrogen peroxide detoxification system of plant cells under abiotic stress.

Detection principle

Ascorbate Peroxidase (APX) can catalyze the reaction between ascorbic acid (ASA) and hydrogen peroxide (H2O2), and ASA can be oxidized to Monodehydroascorbic acid (MDASA). The absorbance of solution at 290 nm will decline as the oxidation of ASA. The APX activity can be calculated by detecting the decrease of A290.

Experimental instrument

Test tube, Micropipettor, Vortex mixer, Centrifuge, Incubator, Spectrophotometry (290 nm)

Extraction of crude enzyme solution

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment.

Tissue homogenate: Add Reagent 1 into the tissue sample according to the ratio of Weight (g): PBS (mL) =1: 10. Homogenized mechanically with a homogenizer in ice-bath, then centrifuge at 10000 g for 10 min. Take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

Operation steps

1. Preheat the Reagent 2 at 37℃ for 1 hour before detection.

2. Preheat the spectrophotometer for 30 min and set the spectrophotometer to zero with double-distilled water at 290 nm with 1 mL slit cuvette.

3. Blank tube: Add 0.1 mL of Reagent 1 into a 2 mL EP tube.

Sample tube: Add 0.1 mL of sample into a 2 mL EP tube.

4. Add 0.7 mL of Reagent 2 and 0.1 mL of Substrate solution into each tube, mix fully.

5. Add 0.1 mL of Reagent 4 (record the time immediately) and mix fully with vortex instrument.

6. Measure the absorbance at 290 nm at 15 second (A1), then incubate the reaction solution at 37℃ and measure the absorbance at 135 second (A2), respectively. △A=A1-A2.

Note: It can be refer to the following operating table.

※ The reaction time should be strictly controlled.

Technical parameters

1. The sensitivity of the kit is 0.071 U/g tissue.

2. The intra-assay CV is 4.8 % and the inter-assay CV is 6.4 %.

3. The recovery of the kit is 96 %.

4. The detection range of the kit is 0.071-47 U/g tissue.

Notes

1. This kit is for research use only.

2. Please progress strictly with operation procedures.

3. Do not use components from different batches of kit.

4. The validity period of kit is 6 months and the expiration date is on the packing box

5. If the value of A1 is more than 2.0, please dilute the sample and then carry the assay.

6. The reaction time should be strictly controlled.

Citations