With just one look at a gel, a scientist sees the need for a system that keeps track of the information and assists in analysis. That need has spawned a market for gel-documentation systems. This article explores a range of opinions about what scientists should seek in such a system.
When asked about the crucial features in gel/blot documentation systems, Kevin McDonald, senior staff scientist at Bio-Rad Laboratories, lists three: automated imaging; broad application flexibility/multiplex capable; and uniformity, if quantification is to be done.
Other experts agree with many of McDonald’s points, and add others. Exploring these opinions and then considering the needs of a particular labs can be used to focus on the features that matter the most.
Easier operation
With automated imaging, says McDonald, “even novice and nonexpert users can acquire quality images.” That comes in handy with the expanding user base.
“Most gel/blot documentation users are not experts,” says Aldrin Gomes, associate professor in the department of neurobiology, physiology, and behavior at the University of California, Davis. “It is important that the platforms offer an easy-to-use and accurate computer program to analyze and edit digital images.”
Beyond being easy for scientists to use, a system should be easy to keep online and working in a lab as needed. As Ira Augenzucker, product manager at Corning Life Sciences, says, one crucial feature of a gel documentation system is that it should be “simple and easy to maintain.”
Broader application
“With the large and growing choice of detection reagents available today, a system needs to be able to accommodate a scientist’s needs over the breadth of imaging applications required in modern experimental protocols,” McDonald explains. “This is of particular importance for Western blots, as multiplex detection gains popularity due to its enabling reduced sample requirements, higher throughput, and more accurate data quantification.”
The growing number of detection methods demands that a documentation system cover the necessary range. “Documentation systems should offer versatility,” Gomes says. “They should be able to image Ponceau S, Coomassie Blue, fluorescence, and/or chemiluminescence.” In the most useful platforms, scientists can multiplex. As Gomes notes, “Two or more fluorescence detection modules that do not show spectral overlap are great for detecting phosphorylated and nonphosphorylated proteins.”
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It’s not just what a system can see, but how much of it. Gomes indicates that versatility also means the “size of blot that could be performed, from very small to very large blots.”
As a scientist considers purchasing a gel/blot documentation system, flexibility should be high on the list. “Today’s lab equipment needs to be versatile, with the capacity for expansion to new applications,” says Lisa Isailovic, vice president of marketing at Azure Biosystems. “A key element in any gel documentation system is the ability to be upgraded to new applications, such as chemiluminescent westerns, visible and NIR westerns, and other fluorescent applications.” She adds, “This takes a system from being primarily a genomics system into the world of proteomics.”
A typical user often does not know what they will require in terms of a gel/blot documentation systems.
Gomes agrees on the value of future flexibility. He likes a system that includes the “ability to upgrade from one imaging platform, such as chemiluminescence, to add another platform, such as fluorescence.” He adds, “A typical user often does not know what they will require in terms of a gel/blot documentation systems.”
In a system that provides chemiluminescence and fluorescence detection modes, Gomes points out that both have upsides and downsides. For example, he explains that fluorescence is capable of multiplexing and provides a better dynamic range than chemiluminescence, and infrared fluorescence provides sensitivity that is comparable or superior to chemiluminescence; but he also lists a string of shortcomings of fluorescence, including that the “variability in signal intensity of fluorescence depends on the source of the probe and company used,” and “the cost of imager can be more expensive than chemiluminescent imagers.”
For chemiluminescence detection, Gomes sees many benefits, including that “the sensitivity of high-sensitivity ECL—enhanced chemiluminescence—reagents is better than the non-IR fluorophores” and that “more laboratories are familiar with chemiluminescent imagers than fluorescent imagers;” but this approach also includes shortcoming, and Gomes notes several: the “use of chemiluminescence on X-ray film gives a small range of detection, signal intensity generally lasts less than a few hours and you cannot multiplex without stripping the blot.”
Quantifying results
In the past, many uses of gels provided qualitative information, but many scientists now use this technology in quantitative applications. “Western blotting is often referred to as a semi-quantitative assay,” McDonald says. “However, generating a reliably quantitative Western blot is highly desirable.”
To collect data from a gel that can be quantified, McDonald says, the image that is captured must provide uniform data across the blot area. “Even normalization, with housekeeping proteins or a total protein will not be of any value if the quantities derived for the normalization or target protein images are not reliably uniform.”
In thinking about the ability to quantify data, scientists should consider how a system collects the information. “The system should offer high sensitivity,” Gomes says. “New CCD cameras offer this sensitivity and high dynamic range.” He adds, “In my opinion X-ray film imaging produces results that are often not reproducible and should not be used for quantification.” Nonetheless, he notes that chemiluminescence and fluorescence, in particular, offer the best dynamic range.
In discussing imaging capabilities, Augenzucker adds that a system should provide the “ability to capture images of multiple types of gels quickly, easily, and in multiple formats—jpg, tiff—for print and publications.” As an example, he says, “We capture any image in under 30 seconds in either bmp, jpg, or tiff formats, and being Windows-based can support an array of printing options, including thermal printers.”
As this overview indicates, today’s gel/blot systems make it easier than ever for scientists to collect data and still allow more advanced capabilities. Such a system allows “the user to require lower amounts of total protein, lower amounts of primary and secondary antibodies, and offer better reproducibility because of improved hardware and software, which results in more accurate quantification,” Gomes says. With those features, scientists can analyze samples at a lower cost—all while gathering higher quality data, and more of it.
Image: Abstract view of bands. Image courtesy of Bio-Rad Laboratories.
Image: Corning scientists using the Axygen Gel Doc System.