Peroxidase (POD) Activity Assay Kit (Guaiacol, Micro Method) - BioReagent

Cat. No.: P1505848

GRADE & PURITY BioReagent

Synonyms

Peroxidase Assay Kit (Microassay)

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Application

Cell Metabolism, Enzyme activity assay

Overview

Peroxidase (EC 1.11.1.7, POD) is a class of heme-containing oxidases found widely in animals, plants, and microorganisms. It catalyzes various oxidation reactions involving hydrogen peroxide and is closely related to numerous physiological and biochemical processes such as respiration, photosynthesis, and auxin oxidation, playing a significant role in redox processes during cell metabolism.

Assay Principle

Peroxidase catalyzes the oxidation of guaiacol by H₂O₂, producing a tawny-brown product (tetraguaiacol). This product has a characteristic absorption peak at 470 nm. The change in absorbance is used to determine peroxidase activity.

P1505848	Component	48T	96T	Storage
P1505848A	Extraction Buffer	70 mL	70 mL×2	2-8℃
P1505848B	Substrate	3 mL	6 mL	2-8℃. Store in the dark.
P1505848C	H₂O₂	0.1 mL	0.2 mL	2-8℃. Store in the dark.

Please check the volume of all components before the experiment. An extra 10% volume is provided for standard curve preparation or pre-experiments

Required Materials Not Provided

Type	Name	Notes
Instrument	Microplate Reader	Capable of measuring absorbance at 470 nm
Consumables	96-well microplate	Standard transparent plate
Reagents	PBS (pH 7.4), Deionized Water	For diluting standards and samples
Others	Homogenizer (for tissue samples), Incubator, Ice Maker, Refrigerated Centrifuge, Adjustable Pipettes and Tips	Using a multichannel pipette is recommended for high-throughput assays

Experimental Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Extraction Buffer	Ready-to-use; equilibrate to RT before use	Store at 4°C
Substrate	Ready-to-use; equilibrate to RT before use	Protect from light during experiment; store at 4°C protected from light
H₂O₂	Ready-to-use; equilibrate to RT before use	Protect from light during experiment; store at 4°C protected from light

2. Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. Keep samples on ice during assay preparation to prevent denaturation and inactivation.

2.1 Animal Tissue

• Rinse tissue with ice-cold PBS, blot dry.

• Weigh about 0.1 g of tissue, add 1 mL of Extraction Buffer.

• Homogenize in an ice bath.

• Centrifuge at 8,000 g, 4°C for 10 min.

• Collect the supernatant into a new tube and keep on ice for assay.

2.2 Plant Tissue

• Rinse tissue with ice-cold PBS, blot dry, and mince thoroughly.

• Weigh about 0.1 g of tissue, add 1 mL of Extraction Buffer.

• For tissues with low fiber content: Homogenize in an ice bath, then centrifuge as in 2.1.

• For fibrous tissues: Sonicate in an ice bath for 5 min (20% power or 200 W, 3s pulse, 7s interval, 30 cycles), then centrifuge as in 2.1.

• Collect supernatant and keep on ice.

2.3 Cells or Bacteria

• Collect 5×10⁶ cells/bacteria.

• Wash with ice-cold PBS, centrifuge at 800 g for 2 min, discard supernatant.

• Add 1 mL Extraction Buffer.

• Sonicate in an ice bath (parameters as in 2.2 fibrous tissues).

• Centrifuge at 8,000 g, 4°C for 10 min.

• Collect supernatant and keep on ice.

2.4 Serum or Plasma

• Assay directly.

3. Assay Procedure

3.1 Microplate Reader Preparation

• Preheat for at least 30 minutes. Set wavelength to 470 nm.

3.2 Working Solution Preparation

• Prepare the Working Solution fresh before use.

• For each well, mix 139 µL Extraction Buffer, 50 µL Substrate, and 1 µL H₂O₂ (total 190 µL per well).

• Prepare a master mix based on the number of samples (+ extra volume).

• Pre-warm the Working Solution at 37°C (mammalian) or 25°C (other species) for at least 10 min.

3.3 Assay Setup

• Add 10 µL of sample to the wells of a 96-well plate.

• Add 190 µL of the pre-warmed Working Solution to each well. Mix thoroughly.

3.4 Absorbance Measurement

• Read the absorbance at 470 nm at 30 seconds (A₁) and 90 seconds (A₂) after adding the Working Solution.

4. Calculation of Results

The following derived and simplified formulas are equivalent.

4.1 Data Processing

• Calculate ΔA = A₂ - A₁

4.2 Sample POD Activity Calculation

(1) Based on Sample Weight:

• Activity Unit (U) Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in A₄₇₀ per minute per gram of tissue per mL of reaction mixture.

• Derivation Formula: POD (U/g fresh weight) = ΔA × V_{total reaction} ÷ (W ÷ V_{total extract} × V_sample) ÷ 0.005 ÷ T

• Simplified Formula: POD (U/g fresh weight) = 4000 × ΔA / W

(2) Based on Cell/Bacteria Count:

• Activity Unit (U) Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in A₄₇₀ per minute per 10⁴ cells or bacteria per mL of reaction mixture.

• Derivation Formula: POD (U/10⁴) = ΔA × V_{total reaction} ÷ (500 ÷ V_{total extract} × V_sample) ÷ 0.005 ÷ T

• Simplified Formula: POD (U/10⁴) = 8 × ΔA

(3) Based on Liquid Volume:

• Activity Unit (U) Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in A₄₇₀ per minute per mL of liquid sample per mL of reaction mixture.

• Derivation Formula: POD (U/mL) = ΔA × V_{total reaction} ÷ V_sample ÷ 0.005 ÷ T

• Simplified Formula: POD (U/mL) = 4000 × ΔA

(4) Based on Protein Concentration:

• Activity Unit (U) Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in A₄₇₀ per minute per mg of tissue protein per mL of reaction mixture.

• Derivation Formula: POD (U/mg prot) = ΔA × V_{total reaction} ÷ (C_pr × V_sample) ÷ 0.005 ÷ T

• Simplified Formula: POD (U/mg prot) = 4000 × ΔA / C_pr

Parameter Definitions:

ΔA: Change in absorbance (A₂ - A₁)

V_{total reaction}: Total reaction volume, 0.2 mL

V_sample: Volume of sample added to well, 0.01 mL

V_{total extract}: Total volume of extraction buffer added, 1 mL

W: Sample weight, g

T: Reaction time, 1 min

C_pr: Sample protein concentration, mg/mL

500: Cell/Bacteria number, in units of 10⁴ (e.g., 5×10⁶ cells = 500 × 10⁴)

5. Results Example

• Sample: 0.121 g mouse kidney.

• Measured: A₁ = 0.152, A₂ = 0.159.

• ΔA = 0.159 - 0.152 = 0.007.

• POD (U/g fresh weight) = 4000 × 0.007 / 0.121 = 231.4 U/g fresh weight

Frequently Asked Questions (FAQ)

Q: What should I do if the sample ΔA is too high or too low?

A: If ΔA > 0.4, the POD activity is too high. Dilute the sample appropriately with Extraction Buffer and re-assay (for plant tissues, a 5-fold dilution is recommended). If ΔA < 0.005, increase the sample amount or extend the reaction time, then re-assay.

Q: What if the ΔA for bacterial and serum samples is too low?

A: POD activity in bacteria and serum is typically low. It is recommended to extend the reaction time.

Precautions

1. It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.

2. For tissue samples, cell samples, and others, results can be normalized between samples by determining the protein concentration. We recommend using the Aladdin B665595 BCA Protein Quantification Kit or the R1491648 Ready-to-Use BCA Protein Quantification Kit.

3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.

4. Biochemical reagents are generally irritating and/or toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, head cover, etc.) throughout the experiment and perform steps in a fume hood or biosafety cabinet when applicable.

5. This product is for research use only. Not for use in diagnostic procedures.

Specifications

Synonyms

Peroxidase Assay Kit (Microassay)

Specifications & Purity

BioReagent

Stability And Storage

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

Storage

Store at 2-8°C, Protected from light

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Chemical and Physical Properties

• Sensitivity: Light-sensitive