Chemiluminescent Western Blot Detection

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

Request sampleDownload Tech Tip

Horseradish peroxidase (HRP) 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.

Which HRP chemiluminescent substrate is right for you?

  Pierce ECL Substrate Pierce ECL Plus Substrate SuperSignal West Pico PLUS Substrate SuperSignal West Dura Substrate SuperSignal West Femto Substrate
Detection level Low to mid picogram Low picogram Low picogram to high femtogram Mid femtogram Low to mid femtogram
Signal duration 0.5–2 hours 5 hours 6–24 hours 24 hours 8 hours
Detection methods X-ray film, CCD imager X-ray film, CCD imager, fluorescence imager X-ray film, CCD imager X-ray film, CCD imager X-ray film, CCD imager
Select when… Target and sample is abundant Target is less abundant, sample is limited, and you need chemifluorescent detection Target is less abundant, sample is limited, and you need more sensitivity than an entry-level ECL substrate provides Target is less abundant, sample is limited, and you are using CCD image capture Target is least abundant, sample is precious, and you need maximum sensitivity
Value to you Low cost; easy to switch from other entry-level ECL substrates Best detection flexibility with chemifluorescent detection option Best value; works for majority of western blots Best signal duration Best sensitivity
  Order now Order now Order now Order now Order now

Fast Western Blotting

SuperSignal substrates are also available in our fast western blot kits which can save up to 4 hours compared to ordinary chemiluminescent western blots! Learn more about Fast Western Blot kits

Alkaline phosphatase (AP) substrates

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.