PART 3 OF A 3-PART SERIES ON RELIABLY QUANTIFYING WESTERN BLOTS
Editor’s note: In part two, you learned some of the risks of using housekeeping proteins as loading controls in Western blots and explored alternatives that allow researchers to normalize their results with confidence. In this final segment, we question a widely held belief among scientists: that X-ray film should be the method of choice for detecting chemiluminescent Western blots.
Chemiluminescence is the most widely used Western blot detection chemistry. And to date, X-ray film has been the most popular technique for blot visualization. Although film has been around for decades, it remains popular with researchers due to its high sensitivity and resolution. The perception of affordability is also a big factor; budget conscious labs often prefer film equipment because they view it to be more affordable than digital imagers. However, using X-ray film comes with several significant yet often overlooked challenges that can hinder researchers seeking to publish their data in reputable journals. Here, we look at these obstacles and how to overcome them.
Too Much Saturation?
One of film’s major limitations is its limited linear dynamic range for light detection; film is easily saturated by chemiluminescent signals from the blot. It’s this ease of signal saturation that gives scientists the idea that film is more sensitive than digital imaging. This range is further narrowed, or compromised altogether, when the film is digitized for analysis.1
Signal saturation remains such a big hurdle for researchers because it’s not always obvious when oversaturation has occurred. When working with weakly expressed proteins, researchers often go with longer exposure times at the expense of oversaturating the stronger expression signals, such as those that come as a result of overabundant housekeeping proteins.
Unfortunately, these challenges tend to fly under the radar—mostly because of a common misconception in the scientific community that film produces the highest-quality data from Western blots. In reality, unless film users conduct their experiments with a deep understanding of the technique’s limitations, this method of detection is an approximation at best. For this reason, publications such as Nature provide very specific guidelines for papers that discourage overexposed film.2
Eliminating Signal Saturation Once and for All
On the bright side, there are ways that researchers can overcome film’s limitations and reduce the chance of signal saturation. One of the most important steps is to validate exposure time for film. Researchers can follow numerous protocols to determine the appropriate film exposure time for their experiments. (For more on this, download the PDF: “Determining the Appropriate Film Exposure Time.”3)
Antibody concentration is another important consideration.4 Accurate quantitation of protein expression is only possible if researchers follow appropriate experimental procedures to determine the linear and quantitative dynamic range for each target protein. A two-fold dilution series of the protein lysate is needed to determine the linear range of quantitation. This is dependent on the abundance of each target or loading control protein in the sample. Ideally, each antibody should first be tested with a dilution series of a pooled protein lysate from the study samples. Doing this will ensure that researchers use the appropriate dilutions of samples for accurate and normalized quantitation of the target proteins through densitometric analysis.
Digital Landscape Expands
Even as X-ray film continues to be a standard option for labs, there are new digital imaging technologies gaining attention in the Western blotting market. These digital imaging systems are affordable, boast a better linear dynamic range than X-ray film, have lower limits of detection and directly address the main challenges of film.
Digital systems are able to capture the less-intense signals that are missed by film, and can do so without compromising stronger signals to saturation.
It’s also worth noting that the costs of digital imaging systems are very comparable to film, especially when factoring in the cost of the developer, ongoing running costs, and the increasing price of film.
Coupled with new reagents, new digital imaging systems give researchers the means to produce solid quantitative Western blot data—as long as the process is carried out with proper technique, validation, and controls. In fact, a recent study4 found that using a two-fold dilution series of protein lysate along with a digital imaging system resulted in a dynamic range nearly an order of magnitude greater than film (0.04-2.5ng versus 0.04-0.31ng) for the protein that they probed for.
A Future of New Options
Western blotting has served a very important role in protein science for the past 30 years, and that is sure to continue. While X-ray film has gotten the job done for researchers so far, it also is prone to challenges—leaving researchers looking for more effective ways to obtain reliable Western blot data. By adopting reproducible technologies such as digital imaging systems, scientists can regain confidence in Western blotting and ensure excellent data quality.
This is the final segment of a three-part series on ensuring integrity in your Western blot data. Read the other parts of this special series on using checkpoints to monitor the progress of your western blots and the pitfalls of using housekeeping proteins as loading controls.
For more information on western blotting methods, ideal transfer conditions, and troubleshooting western blots, visit Western Blotting - Applications and Technologies.
1. Gassman M, Grenacher B, Rohde B, Vogel J. Quantifying Western blots: pitfalls of densitometry. Electrophoresis. 2009; 30(11): 1845-55. doi: 10.1002/elps.200800720
4. Taylor SC, Berkelman T, Yadav G, Hammond M. A Defined Methodology for Reliable Quantification of Western Blot Data. Mol. Biotechnol. 2013. doi: 10.1007/s12033-013-9672-6.