b'Chemiluminescent substrates The choice of substrate for chemiluminescent western blotting is determined by the reporter enzyme that is selected. Specifically, luminol- and acridan-based reagents are chemiluminescent horseradish peroxidase (HRP) substrates. For chemiluminescent detection of alkaline phosphatase (AP), acridan- and 1,2-dioxetanebased substrates are available (Figure 22). HRP substrates generally offer better detection sensitivity and buffer compatibility than AP substrates (Table 17).A BFigure 22. Chemiluminescent reaction of luminol and Invitrogen CDP-Star Substrate. (A) Chemiluminescence is a property of chemical reactions that emit light as a byproduct. Luminol is one of the most widely used chemiluminescent reagents. The oxidation of luminol by peroxide results in the creation of an excited-state product called 3-aminophthalate. This product decays to a lower-energy state by releasing photons of light. (B) Chemiluminescent reaction of CDP-Star Substrate with AP. CDP-Star Substrate is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. Light emission occurs only during the enzymesubstrate reaction.Table 17. Comparison of HRP and AP substrates for western blot detection.HRP substrates AP substratesSensitivityFemtogram sensitivity Picogram sensitivitySignal generation Immediate Gradually increases with signal maximum at ~3060 minutesSignal duration Up to 24 hours 2496 hoursConsiderations Compatible with common buffers such as TBS and PBS Not compatible with phosphate buffersWhen to use Antibodies or probes conjugated to HRP Antibodies or probes conjugated to AP44'