Plant Polyphenol Oxidase (PPO) Activity Assay Kit (Catechol, Micro Method) - BioReagent, high purity

Cat. No.: P1501774

GRADE & PURITY BioReagent

Overview

Polyphenol Oxidase (PPO, EC1.10.3.1) is widely found in the plastids of plants, fungi, and insects. It is a copper-containing oxidase that can oxidize monophenols and diphenols to produce quinones, leading to browning. It is closely related to fruit and vegetable processing, tea quality, and tissue culture. The Plant Polyphenol Oxidase (PPO) Activity Assay Kit (Catechol, Micro method) provides a simple method to detect the activity level of polyphenol oxidase (PPO) in plant tissue samples.

Detection Principle: Polyphenol oxidase (PPO) in the sample catalyzes the oxidation of catechol to produce quinones, which have a characteristic absorption peak at 410 nm. The rate of increase in absorbance at 410 nm is measured to calculate PPO activity.

P1501774	Component	48T	96T	Storage
P1501774A	Extraction Buffer	60 mL	120 mL	2-8℃
P1501774B	ReagentⅠ	24 mL	48 mL	2-8℃
P1501774C	ReagentⅡ	6 mL	12 mL	2-8℃. Store in the dark.

Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.

User-Prepared Instruments and Reagents

• Microplate reader or visible spectrophotometer (capable of measuring absorbance at 410 nm)

• 96-well plate or micro glass cuvettes, adjustable micropipettes and tips

• Refrigerated centrifuge, ice maker, constant temperature water bath

• Deionized water

• Homogenizer

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Notes
Extraction Buffer	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C.
ReagentⅠ	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C.
ReagentⅡ	Ready-to-use; Equilibrate to room temperature before use.	Store at 4°C protected from light; Toxic, handle with care.

2. Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month.

2.1 Preparation of Crude Enzyme Extract

Homogenize the plant sample in ice-cold Extraction Buffer with a mass (g) to volume (mL) ratio of 1:5 to 1:10 (recommended: 0.1 g tissue + 1 mL Extraction Buffer). Centrifuge the homogenate at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.

2.2 Boiled Sample Control

Take an appropriate amount of the crude enzyme extract and heat it in a boiling water bath for 5 minutes (seal to prevent moisture loss). Cool to room temperature.

3. Assay Steps

3.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 410 nm. For spectrophotometers, zero the instrument with deionized water.

3.2 Sample Measurement (Add reagents sequentially into 1.5 mL microcentrifuge tubes):

Reagent	Control Tube (μL)	Test Tube (μL)
Boiled Sample	50	0
Sample (supernatant)	0	50
ReagentⅠ	200	200
ReagentⅡ	50	50

3.3 Mix thoroughly. Incubate in a 25°C water bath for 10 minutes, then immediately transfer to a boiling water bath for 10 minutes. After mixing, centrifuge at 5,000 g, 25°C for 10 minutes. Collect the supernatant. Transfer 200 μL of the supernatant to a micro glass cuvette or 96-well plate. Measure the absorbance of both the Test tube and Control tube at 410 nm. Calculate ΔA = A_test - A_control.

Note:

• Each test tube requires a corresponding control tube.

• It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before the formal experiment.

• The optimal reaction temperature for PPO may vary slightly among different samples and can be adjusted between 25-37°C.

• If the sample absorbance is less than 0.02, consider increasing the sample volume appropriately. If the sample absorbance is greater than 1, it is advisable to dilute the sample before assay.

4. Calculation of Results

Note: We provide both the derived formula and a simplified formula. They are equivalent. It is recommended to use the simplified formula in bold for final calculation.

4.1 Calculation Formula when using a 96-well plate:

Unit Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in absorbance at 410 nm per minute per gram of tissue per mL of reaction mixture.

PPO Activity (U/g fresh weight) = ΔA × V_{total reaction}÷ (W × V_sample / V_{total sample} ) ÷ 0.005 ÷ T = 120 × ΔA / W

4.2 Calculation Formula when using a micro glass cuvette:

Unit Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.01 in absorbance at 410 nm per minute per gram of tissue per mL of reaction mixture.

PPO Activity (U/g fresh weight) = ΔA × V_{total reaction} ÷ (W × V _sample / V_{total sample} ) ÷ 0.01 ÷ T = 60 × ΔA / W

Parameter Definitions:

• V_{total reaction}: Total volume of the reaction system (0.3 mL)

• V_sample : Volume of sample added to the reaction (0.05 mL)

• V_{total sample} : Volume of Extraction Buffer added during homogenization (1 mL)

• T: Reaction time (10 minutes)

• W: Sample weight (g)

Precautions

This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear lab coats and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.