Introduction to Chemiluminescence Western Detection
Chemiluminescent Western blot detection is an indirect, enzymatic method.
- Secondary antibodies are labeled with horseradish peroxidase (HRP), an enzyme reporter
- Chemiluminescent substrate is applied to the blot
- Luminol is oxidized in the presence of HRP and H2O2 to form an excited state product that emits light
- This method is known as “enhanced chemiluminescence”, or ECL
Light produced by the enzymatic reaction is detected by exposure to x-ray film, or by digital imaging with a CCD camera system (such as the Odyssey® Fc) or the C-DiGit® Blot Scanner.
Chemiluminescent detection is very sensitive and widely used, but has several limitations:
- Signal is dynamic and transient, fading exponentially over time
- Multiple exposures are generally required to capture optimal images and avoid signal saturation
- Signals cannot be multiplexed for normalization
- Quantification is challenging, especially if film exposure is used