Molecular Probes® extremely versatile Amplex® Red and Amplex® UltraRed reagents were designed to be highly stable and sensitive substrates for enzyme activity assays, offering:

  • Quantitative enzyme activity detection
  • Extended dynamic range and sensitivity compared to classic colorimetric oxidase assays
  • Fluorescence emission outside the range of compound autofluorescence
  • High-throughput compatibility

Amplex® Red reagent technology

Amplex® Red reagent is a colorless substrate that reacts with hydrogen peroxide (H2O2) with a 1:1 stoichiometry to produce highly fluorescent resorufin (excitation/emission maxima=570/585 nm) (Figure 1). Amplex® Red reaction can be used to routinely detect as little as 10 picomoles of H2O2 in a 100 µL volume (50 nM, Figure 2), at least a 10-fold greater sensitivity than that attained with the commonly used scopoletin assay for hydrogen peroxide. In the scopoletin assay, HRP catalyzes conversion of the fluorescent scopoletin to a nonfluorescent product. Unlike scopoletin, the Amplex® Red reagent is a fluorogenic substrate with very low background fluorescence. Consequently, assays using Amplex® Red as the substrate result in an increase in fluorescence, not a decrease—an inherently superior method for enzymatic assays. Amplex® UltraRed reagent—the second-generation Amplex® substrate—exhibits increased resistance to oxidation and works better in lower pH environments. It is used in assays that have an acid intermediate resulting in a lower reaction pH (i.e., myeloperoxidase using the EnzChek® MPO Activity Assay Kit).

Other advantages of the Amplex® Red reaction over scopoletin-based H2O2 assays include high chemical stability of the Amplex® Red reagent and its fluorescent product, resorufin, and the long-wavelength spectra of resorufin. Because resorufin has excitation/emission maxima of ~570/585 nm (versus 360/460 nm for scopoletin), there is much less interference from autofluorescence in most biological samples.


 
Figure 1. Principle of coupled enzymatic assays using our Amplex® Red reagent. Oxidation of glucose by glucose oxidase results in generation of H2O2, which is coupled to conversion of the Amplex® Red reagent to fluorescent resorufin by HRP.   Figure 2. Detection of H2O2 using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. The inset shows the sensitivity and linearity of the assay at low levels of H2O2.
Request a custom assay

We offer Amplex® Red and Amplex® UltraRed reagents in custom formats:

  • Substitution of Amplex® Red reagent in an existing assay with Amplex® UltraRed reagent
  • Special bulk packaging of our current assays
  • Build your own enzyme-coupled Amplex® UltraRed or Amplex® Red assay

For more information, please contact our Custom Assays Group

Ordering information

Amplex® Red– and Amplex® UltraRed–coupled assays permit the ultrasensitive quantitation of a diverse assortment of analytes, including glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline and acetylcholine, inorganic phosphate and pyrophosphate, as well as hydrogen peroxide. Find these products in the table below or explore our entire range of microplate assays.

Sku Name Size Price Qty
A12217 Amplex® Acetylcholine/Acetlycholinesterase Assay Kit 500 assays USD 266.00
A22180 Amplex® Red Catalase Assay Kit 400 assays USD 286.00
A12216 Amplex® Red Cholesterol Assay Kit 500 assays USD 277.00
A22179 Amplex® Red Galactose/Galactose Oxidase Assay Kit 400 assays USD 275.00
A22189 Amplex® Red Glucose/Glucose Oxidase Assay Kit 500 assays USD 272.00
A12221 Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit 200 assays USD 264.00
A12214 Amplex® Red Monoamine Oxidase Assay Kit 500 assays USD 274.00
A22178 Amplex® Red Neuraminidase (Sialidase) Assay Kit 400 assays USD 270.00
A22188 Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit 500 assays USD 261.00
A12218 Amplex® Red Phosphatidylcholine-Specific Phospholipase C Assay Kit 500 assays USD 285.00
A12219 Amplex® Red Phospholipase D Assay Kit 500 assays USD 268.00
P22061 PiPer™ Phosphate Assay Kit 1,000 assays USD 330.00
P22062 PiPer™ Pyrophosphate Assay Kit 1,000 assays USD 332.00
A12220 Amplex® Red Sphingomyelinase Assay Kit 500 assays USD 270.00
A22181 Amplex® Red Uric Acid/Uricase Assay Kit 400 assays USD 270.00
A22182 Amplex® Red Xanthine/Xanthine Oxidase Assay Kit 400 assays USD 272.00
A12222 Amplex® Red Reagent 5 mg USD 148.00
R363 Resorufin, Sodium Salt - Reference Standard 100 mg USD 64.75
A36006 Amplex® UltraRed Reagent 5 x 1 mg USD 197.00
A33855 Amplex® Red/UltraRed Stop Reagent 1 set USD 83.50
E33856 EnzChek® Myeloperoxidase (MPO) Activity Assay Kit 1 kit USD 499.00