Chemiluminescence is the western blot detection method of choice in many protein laboratories, as it provides high sensitivity and convenience for detection with film or digital imaging equipment. Chemiluminescent substrates are popular because they offer several advantages over other detection methods.
- Allows for multiple exposures to be made in order to obtain the best image
- Blots may reprobed to optimize detection, or to visualize a second protein
- Detects and quantitates a wide range of protein concentrations
- Yields the greatest sensitivity of any available detection method
HRP substrates AP substrates
Chemiluminescent substrates for horseradish peroxidase (HRP) are two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the equal volumes of the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. Although x-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, cooled CCD cameras offer the advantages of qualitative analysis, instant image manipulation, higher sensitivity, greater resolution and a larger dynamic range than film. Plus, you don't have to spend quality time in the darkroom.
We offer five types of chemiluminescent substrates for western blot detection with horseradish peroxidase enzyme (HRP). Use the table below to select the most appropriate HRP Chemiluminescent substrate based on abundance of your target protein of interest, abundance of sample containing the target protein, and the level of sensitivity and type of instrumentation available for detection.
For western blot detection based on alkaline phosphatase (AP), we offer our CDP-Star™ substrate that delivers picogram level sensitivity and is compatible with both traditional x-ray film and CCD-based imaging. Choose from a standalone substrate, Novex™ AP Chemiluminescent Substrate or a complete kit, the WesternBreeze™ Chemiluminescent Immunodetection kit which contains all solutions necessary for your application including blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution, ready-to-use chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter papers, polyester sheet for even substrate development on the membrane.