Peroxidase Assay Kit (Fluorometric, Colorimetric Method)

Cat. No.: A1522298

GRADE & PURITY BioReagent, Suitable for Analysis, Colorimetry

Panoramica

Peroxidase (e.g., Horseradish Peroxidase, HRP) is a ubiquitous oxidoreductase in living organisms that plays critical roles in cellular oxidative stress, energy metabolism, and immune responses. Therefore, peroxidase activity serves as a key indicator for evaluating cellular redox status and metabolic function, with important applications in metabolic research, drug discovery, and clinical diagnostics. Furthermore, due to its high catalytic efficiency and stability, peroxidase is widely used in enzyme-linked immunosorbent assay (ELISA), Western Blot, and biosensor development. Accurate quantification of peroxidase activity is essential for product development and quality control.

This kit utilizes the highly sensitive Amplex Red fluorescent probe to detect peroxidase activity. In the presence of hydrogen peroxide (H₂O₂), peroxidase catalyzes the stoichiometric 1:1 oxidation reaction between Amplex Red and H₂O₂ to produce the strongly fluorescent product resorufin. Sensitive quantification of peroxidase activity is achieved by measuring resorufin fluorescence (excitation at 560 nm, emission at 590 nm) or absorbance at 560 nm (A560). Signal intensity is proportional to peroxidase activity.

The kit supports both absorbance and fluorescence detection, featuring high sensitivity, wide linear range, and low sample consumption. With a sample volume of 50 µL:

• Absorbance assay: good linearity from 0.05 to 0.8 mU/mL
• Fluorescence assay: good linearity from 0.05 to 0.4 mU/mL.

A1522298	Components	Appearance	500 T	1000 T	Storage conditions
A1522298A	Horseradish Peroxidase	Liquid	0.02 mL	0.05 mL	-20℃.dark.
A1522298B	Amplex Red	Liquid	0.5 mL	1.0 mL	-20℃.dark.
A1522298C	H₂O₂ （3%）	Liquid	0.4 mL	0.8 mL	-20℃.dark.
A1522298D	Assay Buffer   (20×)	Liquid	5 mL	10 mL	-20℃.

Usage Method

1. Stock Solution Preparation

1)        Thaw 20× Reaction Buffer, 10 mM Amplex Red Stock, and 50 U/mL HRP on ice; equilibrate to room temperature before use.

2)        Dilute 20× Reaction Buffer with deionized water at 1:19 to prepare 1× Reaction Buffer for use.

3)        10 mM Amplex Red is a ready-to-use liquid stock. It may also be diluted to 1 mM for easier volume calculation.

4)        50 U/mL HRP is a ready-to-use liquid stock. Divide unused enzyme into single‑use aliquots and store at -20°C.

5)        20 mM Hydrogen Peroxide (H₂O₂) Working Solution

Dilute the ~3% H₂O₂ stock with 1× Reaction Buffer to the desired concentration; actual concentration is labeled on the vial.

Example: Add 22 μL of 3% (0.91 M) H₂O₂ to 978 μL of 1× Buffer to obtain 20 mM working solution.

Note: The stabilized 3% H₂O₂ stock degrades slowly, but the 20 mM working solution is unstable and must be used within a few hours of preparation.

2. Standard Preparation (0 - 2.0 mU/mL)

1)        Prepare standards in the range 0-2.0 mU/mL using a 100 mU/mL intermediate stock.

2)        Dilute 50 U/mL HRP with 1× Reaction Buffer to 100 mU/mL intermediate stock.

3)        Prepare standards directly at the following concentrations (direct preparation is more accurate than serial dilution):

0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mU/mL

Dilute all standards with 1× Reaction Buffer.

4)        Use freshly prepared standards for each experiment. Match the diluent for samples and standards to minimize error..

3. Sample Preparation

Fresh samples are recommended. If not assayed immediately, store samples at -80℃ for up to 1 month; thaw on ice before use.

1)        Animal Tissue

Rinse tissue with cold PBS to remove blood; blot dry. Weigh 0.1 g, add 1 mL of cold 1× Reaction Buffer, and homogenize on ice. Centrifuge at 10,000 g, 4℃ for 5 min. Collect supernatant on ice for assay.

2)        Plant Tissue

Weigh ~0.1 g sample, add 1 mL of cold 1× Reaction Buffer, and homogenize. Sonicate on ice for 5 min (20% or 200 W; 3 sec on, 7 sec off; 30 cycles). Centrifuge at 10,000 g, 4℃ for 5 min. Collect supernatant on ice for assay.

3)        Cells / Bacteria

Collect 5×10⁶ cells or bacteria; wash with cold PBS. Add 0.1 mL of cold 1× Reaction Buffer, then homogenize or sonicate on ice for 5 min. Centrifuge at 12,000 g, 4℃ for 5 min. Collect supernatant on ice for assay.

4)        Plasma, Serum, Urine, and Other Biological Fluids

Deproteinize before use:Filter through a 10 kDa ultrafiltration tube and collect filtrate; or

Mix sample : 30% ZnSO₄ solution = 20:1, vortex, centrifuge at 10,000 g, room temperature for 5 min, collect supernatant.

4. Detection Working Solution Preparation

Prepare fresh before use; store on ice and protect from light.

Prepare working solution containing 160 μM Amplex Red + 0.4 mM H₂O₂:

1)        1 mM Amplex Red Stock: 160 μL

2)        20 mM H₂O₂ Working Solution: 20 μL

3)        1× Reaction Buffer: 820 μL

4)        Total volume: 1 mL (enough for ~20 assays); final concentrations are halved in the reaction.

5. Peroxidase Activity Assay

1)        Set up reactions as follows:

Reagent	Standard Wells	Sample Wells
Standard	50 μL	/
Sample	/	50 μL
Detection Working Solution	50 μL	50 μL

2)  Mix thoroughly and incubate at 25℃ for 20-30 min in the dark.

• Absorbance mode: read at 560 nm
• Fluorescence mode: excitation 560 nm, emission 590 nm

6. Data Analysis

1)        Subtract blank (no HRP control) values for standard curve construction.

2) ΔA _Standard = A_Standard− A _Blank 3) 

ΔA _Sample = A _Sample − A _Blank

4) Plot a standard curve with standard concentration on the x‑axis and ΔA on the y‑axis. Calculate peroxidase activity in samples from the curve.

Precautions

1.        Amplex® Red is light‑sensitive and easily oxidized; protect from light throughout and use promptly after opening/preparation.

2.        Excessively high peroxidase concentration reduces signal; dilute samples before assay if needed.

3.        H₂O₂ working solution is unstable; must be prepared fresh.

4.        Avoid reductants such as DTT or 2‑mercaptoethanol in the reaction, which interfere with detection.

5.        Fresh standards and a new standard curve must be used in every experiment.

Specifications

Sinonimi

Peroxidase Assay Kit

Specifiche e purezza

BioReagent,Colorimetry,Suitable for Analysis

Grado

BioReagent, Colorimetry, Suitable for Analysis

Applicazione

Cell Metabolism

Campo di rilevamento

Absorption method 0-0.8mU/mL, fluorescence method 0-0.4mU/mL

Metodo di rilevamento

Fluorometric, Colorimetric Method

Quantitative

Quantitative

Detection instrument

Microplate reader

Detection wavelength

吸光法波长A560；荧光法激发波长560，发射波长590

Sensibilità

Light-sensitive

Detection duration

10 min

Sample volume

50 μL

Stoccaggio e spedizione

Condizioni di conservazione di stoccaggio

Store at -20°C

Spedito in

Ice chest + Ice pads

Stabilità e conservazione

Each component has a shelf life of 1 year under corresponding storage conditions.

Immagini

Cell Metabolism assay: Peroxidase Assay Kit (Fluorometric Absorbance Microassay) (A1522298) - Fluorometric assay

Peroxidase Assay Kit (Fluorometric/Absorbance/Microassay)(A1522298) - Fluorometric assay 

Peroxidase in the sample catalyzes the reaction of hydrogen peroxide (H₂O₂). Under horseradish peroxidase (HRP), Amplex Red reacts quantitatively with H₂O₂ at a 1:1 molar ratio to generate resorufin. Peroxidase activity is sensitively quantified by measuring resorufin fluorescence (excitation at 560 nm, emission at 590 nm) or absorbance at 560 nm (A560), with signal intensity proportional to peroxidase concentration. When the sample volume is 50 µL, the colorimetric assay method exhibits a good linear relationship in the concentration range of 0.05-0.4 mU/mL..

Cell Metabolism assay: Peroxidase Assay Kit (Fluorometric Absorbance Microassay) (A1522298) - Colorimetric assay

Peroxidase Assay Kit (Fluorometric/Absorbance/Microassay)(A1522298) - Colorimetric assay 

Peroxidase in the sample catalyzes the reaction of hydrogen peroxide (H₂O₂). Under horseradish peroxidase (HRP), Amplex Red reacts quantitatively with H₂O₂ at a 1:1 molar ratio to generate resorufin. Peroxidase activity is sensitively quantified by measuring resorufin fluorescence (excitation at 560 nm, emission at 590 nm) or absorbance at 560 nm (A560), with signal intensity proportional to peroxidase concentration. 

When the sample volume is 50 µL, the colorimetric assay method exhibits a good linear relationship in the concentration range of 0.05-0.8 mU/mL..

Application Protocols

Tested applications, recommended dilutions, and step-by-step protocols are not specified for this item; refer to the kit booklet/CoA/Spec Sheet for authoritative instructions.

General best practices (not item-specific):

• Equilibrate all components to room temperature before use unless otherwise directed.
• Prepare fresh working solutions of substrates/standards immediately before assay.
• Run a full standard curve alongside samples on each plate; include duplicates or triplicates.
• For fluorometric mode, set the instrument to the excitation/emission specified in the kit manual (values not provided here). For colorimetry, select the wavelength recommended for the chromophore in this kit (not provided here).
• Record kinetic traces where possible to confirm linear initial rates.

These guidance points are generic; rely on the product’s official protocol for volumes, incubation times, and detection settings.

Biological Roles

This kit measures the catalytic activity of peroxidase-class enzymes.

General background (literature; not item-specific):

• Peroxidases are widespread oxidoreductases that utilize hydrogen peroxide (or organic peroxides) as electron acceptors to oxidize diverse substrates (phenols, amines, halides). Heme peroxidases (e.g., HRP, myeloperoxidase, lactoperoxidase) cycle through high-valent oxo-iron intermediates (Compounds I and II) to effect substrate oxidation.
• Biological significance:
  • In plants, class III peroxidases participate in cell wall remodeling, lignification, and defense responses.
  • In microbes, peroxidases detoxify peroxides and contribute to oxidative stress management.
  • In animals, peroxidases function in host defense and hormone biosynthesis; peroxidase-like activities are also leveraged in biochemical assays.
• Analytical utility:
  • Peroxidase conjugates (e.g., HRP-labeled antibodies) are mainstays in ELISA, blotting, and histochemistry due to high turnover and robust signal generation.

This product is for laboratory research use only and is not intended for diagnostic or clinical use.

Buffer Applications

As an activity assay kit, buffer selection is governed by the kit protocol.

Item-specific buffer formulations and recommended pH range: Not specified for this item; refer to CoA/Spec Sheet.

General guidance (literature; not item-specific):

• Neutral pH buffers such as phosphate (50–100 mM, pH ~7.0–7.4) or HEPES (25–50 mM, pH ~7.2–7.5) commonly support peroxidase activity and substrate stability.
• Avoid components that quench radicals or scavenge H2O2 (e.g., high glutathione, ascorbate, sodium azide) unless studying inhibition.
• Include chelators/detergents only if validated to not inhibit the enzyme or interfere optically. Non-ionic detergents can elevate background in fluorescence assays.
• For fluorometric mode, use black plates and low-autofluorescence buffers; for colorimetric mode, clear plates and buffers with minimal absorbance at the detection wavelength.

Follow the specific kit instructions for buffer composition, ionic strength, and any required cofactors.

Green Alternatives

While this kit is purpose-built, greener practices can be integrated into its use.

• Reduce hazardous solvent use:
  • If any substrate is supplied in an organic solvent (e.g., DMSO), minimize final solvent concentration consistent with assay performance (literature practice ≤1–2% v/v).
• Waste minimization:
  • Opt for microvolume formats (384-well or 1536-well) to cut reagent consumption and waste generation.
  • Use plate sealing to limit volatilization and light exposure, improving stability and reducing repeats.
• Safer chemistry choices (general comparison; not item-specific): | Aspect | Fluorometric mode | Colorimetric mode | | --- | --- | --- | | Sensitivity | Higher; supports miniaturization | Moderate; robust | | Light/solvent sensitivity | Often higher; substrate may be light/solvent sensitive | Typically lower | | Instrument need | Fluorescence reader required | Standard spectrophotometer/plate reader | | Waste profile | Potentially less per data point (miniaturized) | Aqueous waste, simpler segregation |
• Energy and storage:
  • Store at the recommended −20°C. Consolidate freezer space and use insulated shippers (as supplied) to reduce dry ice use during internal transfers.

Exact component composition and associated hazards for this item are not specified; consult the SDS/CoA for best green practice tailoring.

Pharmaceutical Uses

This product is an analytical assay kit for research use only.

• Intended role: Quantitative measurement of peroxidase activity in laboratory settings (e.g., enzyme characterization, conjugate QC, inhibitor screening). It is not an excipient and is not intended for formulation into drug products.
• Pharmacopeial status: Not applicable to a multi-reagent research kit.
• Use in pharmaceutical R&D (general; not item-specific):
  • High-throughput screening of redox enzyme modulators.
  • QC of peroxidase-labeled reagents used in bioanalytical assays.
  • Process analytics for bioconjugation workflows involving HRP.

No clinical, diagnostic, or therapeutic claims are made for this item.

Physical Properties

As a multi-reagent kit, bulk physical constants are not defined for the product as a whole.

Item-specific physico-chemical specifications (examples: substrate extinction coefficient, fluorophore λex/λem, buffer composition, pH, solubility): Not specified for this item; refer to CoA/Spec Sheet.

General literature information (for context; not item-specific):

• Fluorometric peroxidase assays commonly use substrates that yield products with excitation/emission in the visible range (e.g., 520–590 nm), enabling high sensitivity in microplate readers.
• Colorimetric peroxidase assays typically generate absorbance at a visible wavelength (e.g., 370–650 nm depending on substrate), facilitating standard spectrophotometer or plate reader measurements.
• Reaction performance is influenced by buffer pH (often near neutral), ionic strength, and presence of interfering reductants or antioxidants. Exact values for this item are not specified.

Quality and Grades

• Grade/Purity listed: BioReagent, Suitable for Analysis, Colorimetry.
  • BioReagent: Indicates suitability for biological/biochemical experiments with attention to impurity profiles relevant to enzyme assays.
  • Suitable for Analysis: Intended for analytical workflows where reproducibility, signal linearity, and low background are critical.
  • Colorimetry: Components appropriate for reliable absorbance-based quantitation.

What this means in practice:

• Low background signal and controlled reagent quality to ensure linear standard curves and consistent activity readouts across plates/batches.
• Attention to photostability and purity of chromogenic/fluorogenic substrates to minimize non-enzymatic oxidation and drift.
• Kit-level QC typically includes performance tests (e.g., dynamic range, Z′-factor) in manufacturer’s validations; specific QC metrics for this item are not specified; refer to CoA/Spec Sheet.

Stabilizers/Preservatives: Not specified for this item; refer to CoA/Spec Sheet. If present, stabilizers can extend shelf life of enzymes/standards and suppress spontaneous substrate oxidation. Users should verify compatibility with downstream applications (e.g., avoid azide if assaying heme enzymes susceptible to azide inhibition).

Reaction and Applications

This kit quantifies peroxidase activity via dual readouts.

General assay principles (literature; not item-specific):

• Peroxidase catalyzes the H2O2-dependent oxidation of a reduced substrate (fluorogenic or chromogenic). The oxidized product is detected either by fluorescence (high sensitivity) or absorbance (robust, instrument-agnostic).
• Typical applications:
  • Measuring horseradish peroxidase (HRP) activity in labeling, conjugation QC, or immobilized enzyme preparations.
  • Profiling plant, fungal, or mammalian peroxidases (heme and non-heme) in lysates or purified fractions.
  • Monitoring H2O2-generating oxidase reactions by coupling (oxidase → H2O2 → peroxidase substrate turnover).
  • Screening inhibitors/activators of peroxidases or coupled oxidase systems.
• Practical considerations:
  • Maintain consistent temperature (often ambient) and timing to ensure linear initial rates.
  • Calibrate with a provided standard (H2O2 or product standard) to convert signal to activity or concentration; exact standard type not specified for this item.
  • Minimize light exposure for fluorometric substrates; use black, low-binding plates for fluorescence and clear plates for colorimetry.
  • Control for non-enzymatic background by including no-enzyme and no-substrate wells.

Manufacturer Applications: The catalog lists the product as a Peroxidase Assay Kit (Fluorometric, Colorimetric Method); no additional application notes were provided to expand upon.

Reaction Conditions

Item-specific recommended conditions are not provided in the product data; refer to the kit booklet for authoritative instructions.

General conditions for peroxidase activity assays (literature; not item-specific):

• Temperature: Ambient (20–25°C). Maintain constant temperature for kinetic measurements.
• pH: Near neutral (pH 7.0–7.4) is common; verify substrate/enzyme optima.
• Time: End-point readings typically 10–60 min; kinetic mode can capture initial rates within the first 1–10 min, ensuring linearity.
• Plate type: Black, opaque plates for fluorescence; clear, flat-bottom plates for absorbance.
• Light protection: Minimize exposure for fluorogenic substrates; use foil wrapping when necessary.
• Controls: Include blanks (no enzyme), substrate-only, and standard curves (H2O2 or product standard). Exact standard for this kit is not specified.
• Interference management: Avoid high concentrations of antioxidants/reductants, metal chelators, or azide which can inhibit peroxidase or alter signal.

Expected performance metrics (literature): Fluorescence mode generally offers lower LOD and broader dynamic range than colorimetric mode; precise limits, linear ranges, and molar absorptivities/quantum yields are substrate-dependent and not specified for this item.

Safety and Handling

• GHS classification, signal word, pictograms, and H-statements: Not specified for this item; refer to the kit SDS for each component.
• Likely hazards (general literature, not item-specific): Peroxidase activity assays often include hydrogen peroxide standards and organic substrates/fluorophores. These may be oxidizers, irritants, or harmful if swallowed/inhaled. Some components may be supplied in organic solvents (e.g., DMSO) which can facilitate dermal absorption of solutes.
• PPE: Use lab coat, gloves (nitrile recommended), and safety glasses. Handle solutions in a chemical fume hood where volatile solvents or H2O2 are used.
• Incompatibilities (general): Avoid mixing oxidizing standards with strong reducers; segregate acids/bases as appropriate. Protect light-sensitive substrates/fluorophores from illumination.
• First aid (general): In case of skin/eye contact, rinse with water for ≥15 minutes and seek medical advice. If inhaled, move to fresh air. If ingested, do not induce vomiting; seek medical attention. Always follow your institution’s EHS procedures.
• Waste: Collect organic-solvent and dye-containing waste as hazardous; do not dispose to drain. Deactivate residual peroxide before disposal when permitted.
• Always defer to the official SDS and your institutional risk assessment for authoritative safety guidance.

Solvent Selection

This is an assay kit rather than a solvent. Solvent choice is generally limited to the buffers/reconstitution media supplied or recommended by the kit.

General guidance (literature; not item-specific):

• Use the provided assay buffer or a neutral pH phosphate/HEPES buffer to maintain peroxidase activity and substrate stability.
• Avoid organic co-solvents unless explicitly allowed, as they can alter enzyme kinetics and substrate solubility/reactivity. If a fluorogenic substrate is supplied in DMSO, keep final DMSO ≤1–2% v/v to minimize enzyme inhibition (typical literature practice).
• Remove or minimize components that quench radicals or scavenge H2O2 (e.g., strong antioxidants, excess thiols, azide) unless the goal is to measure inhibition.
• For fluorometric mode, ensure the buffer has low intrinsic fluorescence and lacks detergents/dyes that fluoresce in the detection window.

For this specific item, follow the kit booklet for permitted diluents and co-solvent limits. Item-specific solvent/buffer compatibility: Not specified for this item; refer to CoA/Spec Sheet.

Storage and Reconstitution

• Storage conditions (from Product Data): Store at −20°C.
• Shipping (from Product Data): Shipped in ice chest + ice pads to maintain cold chain.
• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Reconstitution: Not specified for this item; refer to the kit booklet/CoA for exact solvents, volumes, and stability after reconstitution.

General guidance (not item-specific):

• Keep light-sensitive substrates protected from light. After first use, aliquot light- and air-sensitive components to minimize freeze–thaw cycles.
• If any components are supplied lyophilized, reconstitute with the recommended buffer or solvent using sterile, low-autofluorescence water/buffer when applicable. Mix gently; avoid vigorous vortexing if foaming affects readings.
• Record the date of reconstitution and store working solutions at recommended temperatures (often 4°C short-term, −20°C longer-term) when permitted by the kit manual.
• Do not refreeze thawed enzyme solutions unless the manual explicitly allows it. Verify stability (e.g., by control standard curves) before critical experiments.

For definitive stability timelines, on-bench working times, and post-reconstitution shelf life, consult the official kit protocol and CoA.

Structure and Identity

This product is a multi-component Peroxidase Assay Kit supporting both fluorometric and colorimetric readouts. It is not a single defined chemical entity.

• SKU: A1522298
• Product type: Enzymatic activity assay kit for peroxidase (dual-mode: fluorometric and colorimetric)
• Molecular formula: Not applicable to a kit containing multiple reagents.
• Molecular weight: Not applicable to a kit; individual components may vary. Not specified for this item; refer to CoA/Spec Sheet.
• SMILES / InChI / InChIKey: Not applicable (multi-component kit).
• Structural features: The kit typically employs a peroxidase substrate that is oxidized in the presence of H2O2 to yield a fluorescent or chromogenic product (general literature). Exact substrate(s) in this item are not specified; refer to CoA/Spec Sheet.
• 2D structural description (general literature): Common peroxidase substrates include arylamines or phenolic ethers that, upon one- or two-electron oxidation by peroxidase/H2O2, form highly conjugated dyes or fluorophores. The kit’s components may also include buffers and calibrators; details not specified for this item.

Synthetic Utility

This is an analytical kit, not a synthetic building block. Synthetic chemistry applications are not directly applicable.

General relevance to synthetic/analytical chemistry (literature; not item-specific):

• Peroxidase-based assays are frequently used to quantify H2O2 formed as a by-product in oxidase-coupled transformations, enabling reaction monitoring and enzyme engineering.
• In chemoenzymatic workflows, peroxidase readouts can report on oxidase, dehydrogenase (via coupled oxidase), or oxidoreductase cascades, assisting optimization of reaction conditions (pH, cosolvents, temperature) without direct chromatographic analysis.
• Colorimetric/fluorometric outputs allow rapid DoE screening to identify solvent tolerance or inhibitory impurities in synthetic streams.

For precise compatibility of this specific kit with organic cosolvents or reaction media, consult the product manual; item-specific tolerances are not specified here.

Target Specificity

Target specificity is not specified for this item; refer to CoA/Spec Sheet.

General note: Peroxidase activity kits typically detect catalytic activity from a range of peroxidase enzymes (e.g., HRP and other heme peroxidases). However, the exact enzyme specificity, inhibitors, and cross-reactivities for this product have not been provided in the product data.