Invitrogen™ Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit

Catalog No.	Quantity
A22188	500 Assays

Catalog No. A22188 Supplier Invitrogen™ Supplier No. A22188

Description

Sensitive, one-step assay to detect hydrogen peroxide (H₂O₂) or peroxidase activity

The Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit contains a sensitive, one-step assay that uses the Amplex™ Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) to detect hydrogen peroxide (H₂O₂) or peroxidase activity. The Amplex™ Red reagent, in combination with horseradish peroxidase (HRP), has been used to detect H₂O₂ released from biological samples, including cells, or generated in enzyme-coupled reactions. Furthermore, Amplex™ Red reagent can be used as an ultrasensitive assay for peroxidase activity when H₂O₂ is in excess. In the presence of peroxidase, the Amplex™ Red reagent reacts with H₂O₂ in a 1:1 stoichiometry to produce the red-fluorescent oxidation product, resorufin. Resorufin has excitation and emission maxima of approximately 571 nm and 585 nm, respectively, and because the extinction coefficient is high (58,000 ± 5,000 cm–¹M–¹), you can perform the assay fluorometrically or spectrophotometrically. This reaction has been used to detect as little as 10 picomoles of H₂O₂ in a 100 μL volume or 1 x 10–5 U/mL of HRP.

Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit Specifications:

• Ex/Em of reaction product resorufin: ∼571/585 nm
• Sensitive one-step assay to detect hydrogen peroxide or peroxidase in vitro
• Can detect as little as 10 picomoles of hydrogen peroxide or 10 μU/mL of horseradish peroxidase activity in a 100 μL assay volume
• Provides sufficient reagents for approximately 500 assays using reaction volumes of 100 μL per assay
• Designed for a 96-well fluorescence or absorbance microplate reader, but can be easily adapted for other volumes, for fluorimeters, and for spectrophotometers

Find More Substrates for Oxidases

Review Substrates for Oxidases, Including Amplex Red Kits—Section 10.5 in the Molecular Probes™ Handbook for more information on these products.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Order Info

Shipping Condition: Room Temperature

Specifications

• Content And Storage: Store in freezer -5°C to -30°C and protect from light.
• Detection Method: Fluorescence
• For Use With (Application): Enzyme and Metabolic Activity
• For Use With (Equipment): Microplate Reader, Spectrophotometer, Fluorometer
• Product Type: Amplex Red Assay Kit
• Dye Type: Amplex Red
• Format: 96-well plate
• Product Line: Amplex
• Quantity: 500 Assays
• Shipping Condition: Room Temperature
• Excitation Wavelength: 571 nm
• Emission Wavelength: 585 nm

Frequently Asked Questions (FAQs)

I'm using an Amplex Red kit, the reagent changes color to pink almost immediately in my own Krebs-Ringer buffer but not in HBSS. Why is this?

The components of Krebs-Ringer buffer (salts) should not cause oxidation of the Amplex reagent (which, in the presence of peroxidase and H₂O₂ oxidizes to resorufin, which is pink in color and fluorescent). Try water alone (the water used to make the Krebs-Ringer buffer). Since Hank's Buffered Saline Solution is typically purchased rather than made in the lab, it likely would not have the same contaminant. Another option is to degas the buffer prior to use to removed dissolved oxygen radicals.

Can I use the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Cat. No. A22188) to measure H2O2 from frozen cell culture supernatant?

We recommend using fresh samples that have not been frozen since H2O2 degrades over time and more readily in diluted form or in solutions that contain metal ions or natural antioxidants. For assaying cell culture supernatant, we would recommend analyzing samples as soon as possible.

Can Amplex Red Assays be performed using cell lysates?

This is not recommended. The presence of endogenous proteases can complicate the assay by degrading the horseradish peroxidase (HRP). Endogenous peroxidases and antioxidants can modify the H₂O₂ required for the reaction, competing with HRP (and catalase) for the substrate.

The Amplex Red Assays are best performed with either purified enzymes or extracted H₂O₂ in a defined buffer system, extracellular solutions or body fluids (media, serum, etc.) that do not exhibit high levels of endogenous protease or oxidase activity and do not contain antioxidants.

Safety and Handling

WARNING: Reproductive Harm - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.