Polyphenol Oxidase (PPO) Assay Kit

(1)

8th Edition, revised in February, 2018

（FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGNOSIS !）

Polyphenol Oxidase (PPO) Assay Kit

Catalog No: E-BC-K259 Method: Colorimetric method Specification: 100 Assays

This manual must be read attentively and completely before using this product.

If you have any problem, please contact our Technical Service Center for help.

Phone: 240-252-7368(USA) Fax: 240-252-7376(USA) Email: [email protected]

Website: www.elabscience.com

Please kindly provide us the lot number (on the outside of the box) of the kit for more efficient service.

(2)

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

Application

This kit can be used to measure polyphenol oxidase (PPO) activity in serum, plasma or tissue samples.

This kit (100 Assays) can detect 50 samples.

Detection significance

PPO(EC1.10.3.1) is widespread in animals, plants, microbes and cultured cells. It is a copper-containing oxidase that oxidizes monophenols and diphenols to produce quinone, resulting in browning, which is closely related to fruit and vegetable processing, tea quality and tissue culture.

Detection principle

PPO catalyzes oxophenic acid to produce quinone which has a specific absorption peak at 525 nm.

Kit components

Extraction solution: Liquid, 100 mL × 1 vial. Store at 4℃

Reagent 1: Liquid, 20 mL × 1 vial. Store at 4℃.

Reagent 2: Liquid, 5 mL × 1 vial. Store at 4℃.

Experimental instruments

Micro quartz cuvette / 96 wells Micro-plate, High-precision transferpettor, Vortex mixer, Centrifuge, Spectrophotometer (525 nm) / Microplate Reader (525 nm), Water bath, Mortar, double distilled water

Operation steps

1. Extraction of PPO crude enzyme solution:

(1) Extraction of PPO in serum, plasma, fruit juice sample: add the appropriate volume of Extraction solution according to the ratio of the volume of sample (mL): volume of Extraction solution (mL) =1: 5~10 (It is recommended to take 0.1 mL of sample, and add 1 mL of Extraction solution). Mechanical homogenate the sample in ice water bath. Centrifuge at 8000 g for 10min at 4℃, then take the supernatant and preserve it on ice for detection.

(2) Extraction of PPO in tissue sample: add the appropriate volume of Extraction solution according to the ratio of Weight (g): Volume of Extraction solution (mL) =1: 5~10 (It is recommended to weigh 0.1 g of tissue, and add 1 mL of Extraction solution). Mechanical homogenate the sample in ice water bath. Centrifuge at 8000 g for 10min at 4℃, then take the supernatant and preserve it on ice for detection.

(3)

(3) Extraction of PPO in bacteria or culture cells: collect the bacteria or culture cells into the centrifuge tube, centrifuge and discard the supernatant. Add Extraction solution into the sediment according to the ratio of bacteria or cells number: volume of Extraction solution (mL)

=500~1000: 1 (it is recommended to add 1 mL of Extraction solution into 5×10⁶ cells), then treat the sample with sonication on ice (power: 20% or 200W, 3 seconds/time, interval for 10 seconds, repeat 30 times). Centrifuge at 8000g for 10 min at 4℃. Take the supernatant and preserve it on ice for detection.

Operation step:

1. Preheat the spectrophotometer or microplate reader for more than 30 min, set the wavelength at 525 nm and set to zero with distilled water.

2. Operation table

Reagent (μL) Sample tube Control tube

Sample 50

Boiled sample 50

Reagent 1 200 200

Reagent 2 50

Double-distilled water 50

Incubate accurately in 37℃ (mammal) or 25℃ (Other species) water bath for 10min. Then incubate accurately in 95℃ for 5 min and cool to room temperature. Centrifuge at 10000g for 10 min at 25℃. Collect 200 μL of the supernatant to a micro quartz cuvette or a 96 well plate. Measure the absorbance of sample and control at 525 nm respectively. Calculate the △A=Asample-Acontrol.

Note: Each sample tube needs to set a control tube. The crude enzyme solution of different samples can be added to different control tubes, then the water bath treatment is carried out for 5 min.

Calculation of results

Calculation formula for detection with quartz cuvette:

1. Calculate of serum, plasma or fruit juice sample:

Definition: 0.01 OD value changed at 525 nm by 1 mL of serum, plasma or fruit juice sample per minute in 1 mL of reation system that is defined as an enzyme activity unit.

PPO activity (U/mL) = △A×Vtotal÷(Vliquid×Vsample÷Vtotal sample)÷0.01÷T=60×△A÷Vliquid

2. Calculate according to concentration of protein:

Definition: 0.01 OD value changed at 525 nm by 1 mg of protein sample per minute in 1 mL of reation system that is defined as an enzyme activity unit.

PPO activity (U/mg prot) = △A×Vtotal÷(Vsample×Cpr)÷0.01÷T=60×△A÷Cpr 3. Calculate according to the fresh weight of sample:

Definition: 0.01 OD value changed at 525 nm by 1 g of tissue sample per minute in 1 mL of reation system that is defined as an enzyme activity unit.

PPO activity (U/g fresh weight) = △A×Vtotal÷(W×Vsample÷Vtotal sample)÷0.01÷T=60×△A÷W

(4)

4. Calculate according to the density of bacteria or cells:

Definition: 0.01 OD value changed at 525 nm by 1×10⁴ of bacteria or cells per minute in 1 mL of reation system that is defined as an enzyme activity unit.

PPO activity (U/10⁴ cell) = △A×Vtotal÷(500×Vsample÷÷Vtotal sample)÷0.01÷T=0.12×△A

V total: the total volume of the reaction system, 0.3 mL;

V liquid: the volume of the added serum/plasma/ fruit juice sample, 0.1 mL;

V sample: the volume of sample added into the reaction system, 0.05 mL;

V total sample: the volume of the added extract solution, 1 mL;

T: reaction time, 10 min;

W: weight of the sample, g;

Cpr: concentration of protein in sample, mg/mL;

500: the number of bacteria or cells, 5×10⁶.

Calculation formula for detection with Microplate:

1. Calculate of serum, plasma or fruit juice sample:

Definition: 0.005 OD value changed at 525 nm by 1 mL of serum, plasma or fruit juice sample per minute in 1 mL of reation system that is defined as an enzyme activity unit.

PPO activity (U/mL) = △A×Vtotal÷(Vliquid×Vsample÷Vtotal sample)÷0.005÷T=120×△A÷Vliquid

2. Calculate according to concentration of protein:

Definition: 0.005 OD value changed at 525 nm by 1 mg of protein sample per minute in 1 mL of reation system that is defined as an enzyme activity unit.

PPO activity (U/mg prot) = △A×Vtotal÷(Vsample×Cpr)÷0.005÷T=120×△A÷Cpr 3. Calculate according to the fresh weight of sample:

Definition: 0.005 OD value changed at 525 nm by 1 g of tissue sample per minute in 1 mL of reation system that is defined as an enzyme activity unit.

PPO activity (U/g fresh weight) = △A×Vtotal÷(W×Vsample÷Vtotal sample)÷0.005÷T=120×△A÷W 4. Calculate according to the density of bacteria or cells:

Definition: 0.005 OD value changed at 525 nm by 1×10⁴ of bacteria or cells per minute in 1 mL of reation system that is defined as an enzyme activity unit.

PPO activity (U/10⁴ cell) = △A×Vtotal÷(500×Vsample÷÷Vtotal sample)÷0.005÷T=0.24×△A

V total: the total volume of reaction system, 0.3 mL;

V liquid: the volume of the addedserum/plasma/ fruit juice sample, 0.1 mL;

V sample: the volume of sample added into the reaction system, 0.05 mL;

V total sample: the volume of the added extract solution, 1 mL;

T: reaction time, 10 min;

W: weight of the sample, g;

Cpr: concentration of protein in sample, mg/mL;

500: the number of bacteria or cells, 5×10⁶.

(5)

Notes

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 3 months.

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

5. If the activity of PPO is calculated by protein concentration, the protein concentration of the sample needs to be determined separately (E-BC-K318, E-BC-K168, E-BC-K165).