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

Assay uses AmplexRed reagent (10-acetyl-3,7-dihydroxyphenoxazine) to detect hydrogen peroxide (H₂O₂) or peroxidase activity in vitro

. AmplexRed reagent, in combination with horseradish peroxidase (HRP), can detect H₂O₂ released from biological samples, including cells, or generated in enzyme-coupled reactions. AmplexRed reagent can also be used as an ultrasensitive assay for peroxidase activity when H₂O₂ is in excess. In the presence of peroxidase, AmplexRed 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 ± 5000cm–¹M–¹), the assay can be performed fluorometrically or spectrophotometrically. This reaction has been used to detect as little as 10 picomoles of H₂O₂ in a 100μL volume or 1 x 10–5U/mL of HRP.

• Can detect as little as 10 picomoles of hydrogen peroxide or 10μU/mL of horseradish peroxidase activity in a 100μL assay volume
• Provides sufficient reagents for approximately 500 assays using reaction volumes of 100μL per assay

Cell Analysis, Cell Culture, Cell Viability, Proliferation and Function, Enzyme and Protein Activity Assays, Nitro-Oxidative Stress, Oxidase and Peroxidase Activity, Proteins, Expression, Isolation and Analysis, Reactive Oxygen Species, Stem Cell Analysis, Stem Cell Labeling and Detection, Stem Cell Research

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): Hydrogen Peroxidase Assay
• For Use With (Equipment): Microplate Reader, Spectrophotometer, Fluorometer
• Product Type: Amplex Red Assay Kit
• Dye Type: Other Label(s) or Dye(s)
• Format: Tube(s), 96-well plate
• Product Line: Amplex
• Quantity: 500 Assays
• Shipping Condition: Room Temperature

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.