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Key variables affect the analysis of chemiluminescent Western blots

Substrate availability affects signal intensity across the blot

Substrate availability affects signal intensity across the blot. The same samples were loaded 3 times on a single blot, and detected with SuperSignal® West Pico substrate. Non-uniform distribution of substrate made some bands stronger than others (yellow circle).

Detection chemistry and enzyme/substrate kinetics

The enzymatic nature of chemiluminescent detection is a source of error.

  • Signal intensity is dynamic and transient. Light intensity peaks quickly, and fades exponentially with time as the chemical reaction slows. Signal stability depends on the substrate used.
  • Substrate availability and exhaustion affect the chemical reaction. Distribution and accessibility of substrate are not uniform across the blot. Strong bands with high local concentration of HRP enzyme may rapidly deplete the substrate.
  • Signal intensity may not be proportional to the amount of target, or its proportionality may change over time.

These inherent limitations affect signal output, regardless of the method used for signal capture (film, digital imaging, etc).

Signal capture and densitometry

Film is widely used to document chemiluminescent blots, and is very sensitive. However, the response of film to light is non-linear and may introduce error.

  • When film and densitometry are used, accuracy is dependent on factors such as the sensitivity, linear response range, and exposure time of the film.
  • Multiple film exposures are typically required. A single exposure only provides optimal detection across a narrow linear response range — and the linear range is different for every exposure.
  • Faint and strong bands are under-represented on film, and saturation of signals is common.
  • Digitization of films for densitometry can introduce variability. Desktop scanners typically provide limited dynamic range and uneven illumination, and automatic gain controls may drastically affect densitometry results.1

Digital imaging bypasses these limitations, and offers a much wider linear response range than film. However, the enzymatic detection method remains a source of variability.2,3

1. Gassmann et al. Quantifying Western blots: pitfalls of densitometry. Electrophoresis 30:1845-55 (2009).

2. Whitepaper: Chemiluminescent Westerns: how film and photochemistry affect experimental results

3. Whitepaper: Western blot analysis: comparison of film and the C-DiGit® Blot Scanner

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