Chemiluminescent Western Detection Overview

Chemiluminescent Western detection (otherwise known as enhanced chemiluminescence or ECL) is a sensitive, indirect Western blot detection method based upon an enzymatic reaction.

In chemiluminescent Western detection, secondary antibodies are labeled with an enzyme reporter. Typically, this enzyme reporter is horseradish peroxidase (HRP).

After incubation with primary and HRP-conjugated secondary antibodies, the blot is incubated with a luminol-based chemiluminescent substrate. This substrate is oxidized in the presence of the HRP enzyme, which causes the substrate to transiently produce light. This light is then detected by exposure to x-ray film or by digital imaging (such as the Odyssey® Fc Imager or the C-DiGit® Blot Scanner).

What to Know When Considering Chemiluminescence

While chemiluminescent detection is sensitive and widely used, there are some factors to keep in mind when considering chemiluminescent detection.

  • Transient enzymatic reaction
  • Multiple exposures
  • Signals cannot be multiplexed
  • Precise quantification is challenging

LI-COR can help mitigate the effect of some of these factors by moving to digital detection. However, not all of them can be eliminated without transitioning to infrared based detection.

Read more in this whitepaper.

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Considerations When Using Film

The photographic emulsion on x-ray film contains light-sensitive silver halide crystals, called “grains.” Photons of light activate the silver grains and create a latent image. During film processing, activated silver grains are reduced to black metallic silver to create a visible image. Optical density (OD) is the concentration of metallic silver that remains on the developed film.

Film Is Governed by the Reciprocity Law

Film’s response to light is proportional to the intensity of the light and the exposure time. This is known as the reciprocity law. This law only applies across a limited linear response range. Strong and faint signals display “reciprocity failure,” where film response is no longer proportional to exposure.

Strong Signals Can Overpower Film

Strong signals will saturate film. Saturation occurs when all silver grains have been activated, and no more signal can be recorded.

As more silver grains are activated, signal begins to plateau. This is because each new photon of light is statistically less likely to strike an inactive grain.

Film’s response begins to plateau well before saturation is reached. As a result, strong signals are under-represented and often appear similarly dark when compared to fainter signals.

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Figure 1. Saturation of strong signals

Faint Signals May Be Missed

Each silver grain must absorb multiple photons of light to form a latent image. Faint bands may be unable to form a stable latent image, even with long exposures. In a dilution series, this causes signal to drop off abruptly rather than decrease gradually.

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Figure 2. Unresponsiveness to faint signals.

Photographic Emulsion May Affect Image Quality

On film, strong signals blur and spread — obscuring adjacent bands. This “blow-out“ of strong bands makes it difficult to detect both strong and faint signals on the same blot.

Parallax effects (caused by double-emulsion film) also reduce image clarity and sharpness. Scratching and static electricity may cause image artifacts.

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Figure 3. Spreading of strong bands

Read more in this whitepaper.

Variables in Chemiluminescent Analysis

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.).

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Figure 4. 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).

Signal Capture and Densitometry

Film is widely used to document chemiluminescent blots and is 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 signals are under-represented on film, and saturation of signals is common.
  • Digitization of film for densitometry can introduce variability. Office 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

Read more in this whitepaper.

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