We’ve all been there. Sat in front of our PI, staring at a large pile of films, desperately wishing we’d annotated them properly as they came out of the film processor. To avoid these awkward situations and, more importantly, to ensure western blot data sources can be traced and experimental findings reproduced, many researchers are choosing to integrate a protein gel documentation system into their lab. But what advantages do these instruments offer, and what should you look for when choosing one to support your research?

Digital imaging offers numerous benefits

“One major advantage of digital imagers is that they improve the accuracy of image analysis,” notes Jeff Harford, senior product marketing manager at LI-COR Biosciences. “Film contains a limited amount of information, so you can really only perform densitometry. Digital images have a far greater depth of information and you can instead analyze the pixel-by-pixel intensities. This allows you to be more accurate in the overall evaluation of your results, especially if you are using software that is designed specifically for western blot analysis.”

Lindsey Kirby, product specialist at Syngene, adds that a further benefit of digital imagers is that they provide a wider linear dynamic range than film, typically around 4.8 orders of magnitude, making it easier to detect both bright and weak bands on the same blot simultaneously. “Using film, multiple exposures of differing length are often necessary, adding time to workflows while increasing costs and the use of harmful chemicals,” she says. “In contrast, digital imagers usually require just a single acquisition to provide quantitative rather than qualitative data.” According to Paul Liu, Ph.D., product manager, protein quantitation at Bio-Rad Laboratories, another reason to consider using a digital imaging system is that it can allow researchers to multiplex their western blots by switching from chemiluminescence to fluorescent detection. “X-ray film is by nature only capable of capturing a single protein signal,” he says. “Moreover, this results from a dynamic, enzymatic reaction that can produce variable results day-to-day. Fluorescent western blotting uses antibodies labeled with different fluorophores to distinguish protein targets, providing more information from sample material as well as improving experimental reproducibility.” Additional advantages of digital imaging include workflow convenience; a permanent tamper-proof record of any results; and simpler data sharing with colleagues and/or CFR-compliant tools and processes.

Advantages of digital imaging:

  • More accurate image analysis
  • Wider linear dynamic range
  • Saves time
  • No costly consumables (film)
  • Fewer harmful chemicals
  • Quantitative rather than qualitative data
  • Supports fluorescent detection for multiplexing and improved reproducibility
  • Workflow convenience
  • Permanent, tamper-proof record of results
  • Promotes data-sharing

What to look for

So what are the main features you should investigate when choosing a protein gel documentation system? Top of the list is sensitivity, with one way of assuring this being to identify a platform that offers detection of multiple western blot chemistries. “While many researchers decide to purchase a protein gel documentation system to facilitate a move to multiplex fluorescence detection, a platform that also supports chemiluminescent western blotting can offer that extra sensitivity that is sometimes required,” reports Martin Miguez, PharmD, product manager at Azure Biosystems. “Most chemiluminescent western blots are readily adapted to fluorescence detection, increasing the experimental flexibility and improving the workflow and costs, while the ECL is especially useful to support detection of extremely low abundance targets.”

Taking the discussion around sensitivity one step further, Liu observes that although acquisition speed is often used as a proxy for sensitivity, it can be wise to choose a digital imager able to detect a signal regardless of acquisition time. “In some cases, manufacturers sacrifice ultimate sensitivity for speed of acquisition,” he says. “However, a few extra seconds spent capturing an image is small relative to the western blotting workflow.” A further way of ensuring sensitivity is to choose a system that can be easily upgraded, for instance allowing researchers to leverage the advantages of new fluorophores with exceptionally large Stokes shifts as these become available.

Uniformity of illumination and detection are also critical factors in platform selection. “Problems can occur when certain areas of the visual field are brighter than others,” says Liu. “Because western blot experiments are often set up to compare protein expression in one lane versus another, for instance assessing several treated samples against a control run at one side of the gel, the entire field of view must be uniform for results to be accurate.” Harford notes that not all systems are created equal in this regard, emphasizing that non-uniform illumination and detection can lead to results with a considerably higher CV%. Seeing proof of uniformity during instrument selection can help to mitigate this risk.

Another important factor to consider is ease of use. April Wang, application scientist at Analytik Jena, notes that a system providing one-button image capture can significantly reduce user-to-user variability. “An intuitive platform can help to ensure consistency from one experiment to the next,” she says. “Features to look for include automatic tracking of capture and analysis conditions, and in-built editing, annotation, and analysis tools. Also, is the imager small enough to be placed on a standard laboratory bench and does it come with full application and training support? The more easily a digital imager fits into existing workflows, the more likely it is to see uptake.”

A further consideration concerns the instrument software and whether it is aligned with publisher best practices. “While researchers often think ‘It’s just a western blot’, publishers such as JBC have done a great job of highlighting ways for obtaining the highest quality, most replicable western blot data,” says Harford. “The problem is that traditional software packages will simply assign signal values, leaving a lot of work in the hands of researchers when it comes to analysis. By developing our latest software to be in alignment with publisher best practices, we’ve found that the CV% variation between users of different experience levels can be less than 3% compared to >25% with traditional software.”

Although choosing a protein gel documentation system is not a decision that can be rushed, the steady stream of researchers leaving dark rooms around the world is testament to the many advantages these platforms offer. By taking the time to identify and invest in a system that best meets your needs, you can be confident of generating robust, reproducible western blot data for years to come.

Factors to consider when choosing a protein gel documentation system:

  • Sensitivity
  • Dynamic range
  • Uniformity of illumination and detection
  • Types of detection supported
  • Ease of use
  • Size of instrument
  • Software alignment with best practices
  • Readily upgraded
  • Cost of ownership