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Signal intensity peaks and fades as the reaction slows over time.Substrate can be depleted by strong bands with high enzyme concentration.Substrate type affects signal stability due to differing reaction rates.Substrate availability affects signal intensity.While chemiluminescent detection is widely used, there are certain aspects to keep in mind when considering it as a Western blotting method.
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The light photons are then measured with one of two detection methods: x-ray film or digital imaging.įor more information, check out the “Introduction to Western Blotting” learning path in the Lambda U ® education portal. Consequently, HRP causes the substrate to oxidize and transiently produce a signal in the presence of hydrogen peroxide (H 2O 2). The most common chemiluminescent detection substrate, HRP, is luminol-based. The blot is then incubated with substrate to generate a signal, or a production of light. For its enzyme, ECL typically uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). The secondary antibody, which is labeled with an enzyme, binds to the primary antibody. How it WorksĬhemiluminescent Western detection begins when the primary antibody first recognizes the target protein or antigen on the membrane. A distinguishing characteristic is the use of an enzymatic reaction for protein detection. Enhanced chemiluminescence (ECL) or chemiluminescent Western detection is a traditional method for conducting Western blotting research.