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Introduction to Chemiluminescence Western Detection

diagram_ChemiSubstrate

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


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