Understand variables that X-ray film detection introduces into Westerns

Understanding the variables that X-ray film detection introduces into your Western blot experiment is important; they can influence the sensitivity, accuracy, and interpretation of your data. Make sure you are informed of potential issues that can arise with X-ray film and how they can affect your data.

**Figure 1. Left:** A strong signal quickly activates the majority of silver grains in that region. Film response slows and eventually saturates as new photons of light fail to activate new silver grains. **Right:** Faint bands are not effectively detected. When the rate of light emission is too low to activate silver grains, the signals cannot be detected - even with long exposures.

X-ray film’s response to light is not linear and requires multiple exposures to capture your image. The dynamic range, or the span of signal intensities that the detection system detects in a single capture, is also limited. Consequently, signals outside the dynamic range cannot be accurately detected or can go completely undetected by film, which results in a loss of very strong or very faint bands.

Additionally, strong bands tend to blur and spread on film, which makes it difficult to quantify. When stronger bands are located next to faint bands, it becomes even more challenging: longer exposures are required to detect faint bands but cause stronger bands to spread and obscure adjacent or faint bands. This can cause issues in imaging and accurately interpreting your experimental results.

**Figure 2.** On film, strong signals blur and spread to obscure adjacent bands. ARNO protein was detected in cell lysates, using ECL Plus substrate and 90-sec film exposure. Spreading of bands is especially problematic when longer exposures are required to detect faint bands (right side of blot).

There are other options than x-ray film. For example, digital imagers like the Odyssey® Family Imaging Systems can capture all your data in a single image and have a broader dynamic range to include very weak and strong bands in one single image acquisition. Moreover, digital imaging completely eliminates the expenses of a darkroom, film, and developing reagents.

Don’t let X-ray film negatively affect your data. Learn more about the Odyssey Imaging Systems.

Limitations of X-ray Film