Additionally, its wider dynamic range can collect more data and therefore decrease the loss of very strong and weak bands. Digital imaging is useful because it captures more data than x-ray film in a single exposure and reduces the need for multiple exposures. LI-COR can help bypass any potential x-ray film constraints by facilitating a transition to digital imaging. It is therefore important to understand the variability of ECL in its entirety before proceeding with experimentation. Overall, these factors make it difficult to achieve precise quantification. Additionally, chemiluminescence is unable to multiplex signals or simultaneously detect two targets with the same molecular weight in a single sample that is on the same blot. This makes it challenging to accurately quantify the protein abundance on the membrane. Ultimately, it can be difficult to capture the enzymatic reaction for each target simultaneously and at the same point each time.
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.