The peroxidase-catalyzed oxidation of luminol produces a weak flash of light at 425 nm. The incorporation of a so-called enhancer into the buffer forces the flash signal into a glow and greatly improves the analytical characteristics of the reaction in terms of increased signal intensity and duration. Typical enhancer compounds are substituted phenols, the most popular being p-iodophenol and p-hydroxycoumaric acid. The enhanced, HRP catalyzed oxidation of luminol is a complex, multi-step reaction. While enhancers are useful in improving enzyme turnover and increasing the equilibrium concentration of a key intermediate, the luminol radical anion, recent work has shown that, by addition of a suitable catalyst, a further, large increase in light output is obtained:
As a result of this breakthrough, the family of AppliChem's CheLuminate-HRP chemiluminescent substrates has been developed, with specific formulations to meet the various requirements for immunoblotting:
• Outstanding Sensitivity - The intense light output generated by the CheLuminate-HRP substrates translates into a corresponding improvement in sensitivity. Even the economical CheLuminate-HRP PicoDetect Extended formulation provides a sensitivity level at least eight times higher than with phenolic enhancers, down to the low picogram (10-12) range. Detection limits are lowered further with CheLuminate-HRP FemtoDetect, to high-femtogram (10-13), while the ultimate level in sensitivity, is achieved with CheLuminate-HRP FemtoDetect Plus, which is designed to provide femtogram (10-15) - level detection.
• Long Signal Duration - All substrates exhibit long light emission. However, this feature has been specifically maximized in the formulation developed for the CheLuminate-HRP FemtoDetect substrate, which offers a 12-hour light emission – at least ten times longer than with standard phenolic enhancer substrates.
• Versatile Signal Detection - The chemiluminescent signal used in CheLuminate-HRP substrates can be detected either by film or by digital cameras based on CCD sensors. The latter technology offers the advantages of direct image acquisition and manipulation, higher sensitivity, greater resolution and a larger dynamic range.
• Safer - All chemicals used in CheLuminate-HRP substrates were carefully selected for safety. None of the components used in CheLuminate-HRP formulations has been reported to be hazardous to human health.
• More Stable - The shelf life of all CheLuminate-HRP stock reagents exceeds 1 year at 2-8°C. The working solutions, obtained by mixing the two components of the kits in a one-to-one ratio, are stable for either 7 days (CheLuminate PicoDetect), or 24 hours (CheLuminate-HRP PicoDetect Extended, FemtoDetect, FemtoDetect Plus) at room temperature.