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