Although most researchers have used ﬁlm to document Western blots, many may be unfamiliar with the photochemical process that creates a visible image on a sheet of x-ray ﬁlm. Because this process affects data output, it is important to understand how chemiluminescent signals are recorded by ﬁlm – particularly if the results will be quantiﬁed by densitometry.1
What happens when you expose a Western blot to film?
X-ray film is coated with a photographic emulsion that contains light-sensitive silver grains. Photons of light from the chemiluminescent reaction activate individual silver grains, which are then converted to black metallic silver to create a visible film image. Within the film’s linear response range, your results are proportional to light intensity and duration; this is called the Reciprocity Law.
What goes wrong during film exposure?
Film’s linear response range is extremely narrow (1.0 – 1.5 logs). Above and below that narrow range, “reciprocity failure” occurs – and your bands won’t be proportional to the light produced by the chemiluminescent reaction. It’s important to know that both strong and faint signals are not accurately detected by film, which compromises the accuracy of your densitometry results.
How does reciprocity failure affect your densitometry and data analysis?
In this example, film response is only linear between 0.1 ng and 1.56 ng. Above 1.56 ng, bands visually appear stronger on film, but signals are not accurately recorded due to high intensity reciprocity failure.
Improve the accuracy of your results
The photochemistry of film causes a non-linear response of film to faint and strong signals (reciprocity failure). Saturation of strong signals and under-representation of faint signals means that accurate densitometry is severely limited by film’s shortcomings. When you switch to a digital imager, you will get more accurate results. Read the full paper to learn about all the variables that affect accurate quantification:
- Enzyme/substrate kinetics and changes in substrate availability
- Limitations of film exposure and digitization methods
- Difficulty determining the saturation point of strong signals
Read the full study: Chemiluminescent Westerns: How film and photochemistry affect experimental results
1. Baskin, DG and WL Stahl. Fundamentals of quantitative autoradiography by computer densitometry for in situ hybridization, with emphasis on 33P.