Although researchers have for many years relied on film for Western blot analysis, it is becoming increasingly common to find laboratories equipped with sophisticated, film-free Western blot documentation systems. These afford higher sensitivity and a broader linear dynamic range, providing greater experimental reproducibility and making it simpler than ever before for researchers to produce publication-ready images. To meet demands for faster, smarter Western blot analysis, documentation systems are being equipped with a growing range of innovative features, enhancing further still the power and versatility of these instruments.
There is no more sensitive, convenient, or space-efficient way to capture the blot signal...
Kevin McDonald, senior staff scientist at Bio-Rad Laboratories, notes that while Westerns have historically been visualized by developing colored substrates from enzymatic reactions, with the membrane subsequently being taped into a laboratory notebook, Western blot imaging has now become digitalized. “As imaging capabilities have improved over the last two decades and costs have come down, a digital imaging system has become a laboratory requirement rather than a nice-to-have,” he says. “There is no more sensitive, convenient, or space-efficient way to capture the blot signal and taking one image or twenty costs the same.”
One researcher who fully appreciates the utility of Western blot documentation systems is Amy Emery, research associate at the University of Cambridge, who runs Westerns on a regular basis during her work within cancer research. “I have access to a LI-COR Odyssey, which can be used with different combinations of fluorescently labeled secondary antibodies during multiplexing experiments,” she explains. “The Odyssey eliminates the need to produce numerous exposures to film in the dark room, instead letting you simply add your membrane to the instrument and begin scanning. Not only does this help to eliminate user-bias, but at the end of the scan the bands can be quantified. This is particularly useful for normalizing data, for example when comparing levels of a phosphorylated protein to total protein levels across different samples.”
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LI-COR’s digital imagers are referenced in a recent publication by Ghosh et al, within which the authors recommend that wherever possible systems such as these are used instead of film for Western blot detection. Jeff Harford, senior product marketing manager, elaborates that film-based detection and imaging are major causes of Western blot inaccuracies. “Since chemiluminescence is a dynamic reaction impacted by many variables, the use of film makes reproducibility challenging and represents a significant cause of inter-operator variability. Factors including substrate sensitivity, film age, and exposure time can all combine to cause doubt over the relevance of the data.”
To address these issues, LI-COR developed the Odyssey® CLx, a digital imager relying on near-infrared fluorescence. “This is a second generation of the original system we launched in 2001,” says Harford, “offering six logs of linear dynamic range and allowing both strong and faint bands to be seen simultaneously. For those researchers who prefer to use chemiluminescent detection, but wish to achieve this digitally, we developed our Odyssey Fc. This lets one assess where linearity of a target is lost, a process which is extremely difficult with film.” The company also offers an affordable, high-performance chemiluminescence imaging system known as C-DiGit, which, like all of LI-COR’s digital imagers, is supplied with specialized software that permits storage of the raw data without alteration.
With a strong focus on providing multi-functional imaging platforms, Azure Biosystems developed their cSeries imagers so that researchers could perform every stage of their Western blot documentation on a single instrument. Available in several different configurations, the cSeries is suitable for documenting protein and DNA gels, as well as for imaging chemiluminescent, near-infrared, and visible fluorescent Western blots. “We were the first, and are still the only, company to put lasers into a high-resolution CCD-based imaging system that also can image UV gels and visible fluorescence,” says Lisa Isailovic, vice president of marketing. “Lasers offer better sensitivity than LEDs or white light sources, along with faster image capture and real-time detection.”
To complement the cSeries instruments, Azure recently launched their Sapphire Biomolecular Imager, which has the additional capability of phosphor imaging as well as a higher resolution than the cSeries. “This is the only system currently on the market that offers phosphor imaging, laser-based visible to NIR fluorescence and chemiluminescent imaging within one platform,” adds Isailovic, “and it’s compatible with more demanding applications, such as in-cell Western blots and complex 2D gels. Furthermore, similarly to our cSeries instruments, it has been fully integrated with our Sapphire Capture and AzureSpot software programs, which greatly simplify quantitation and improve reproducibility by automating key steps such as band identification and background subtraction.”
Also offering a versatile range of Western blot documentation systems, including the capacity to image chemiluminescent or fluorescent Western blots in addition to Coomassie blue or silver-stained proteins on acrylamide gels, Syngene’s products benefit from application-driven image-capture software. “Our GeneSys software automatically selects the optimal filter and lighting conditions to produce clear images of complex multiplex gels, including low-abundance proteins,” explains Lindsey Kirby, product manager. “Developed for use with our GeneGnome and G:BOX Western blot imagers, GeneSys is equipped with QuickQuant, a feature that allows researchers to perform band quantification down to nanogram levels while the imager is still in use.”
The GeneGnome is a compact benchtop instrument dedicated to chemiluminescence, while the G:BOX imagers range from an entry-level system providing chemiluminescent and fluorescent detection, through to instruments that promise greater resolution and enhanced sensitivity. “The development of high-resolution cameras and high-intensity colored LEDs has been pivotal to the evolution of imaging technology,” adds Kirby. “We’re always looking at what new advances there are with lens, camera, and lighting technologies to improve these systems yet further.”
Currently providing three different ChemiDoc™ imaging systems targeted to the Western blot market, Bio-Rad has capitalized on the benefits of stain-free technology. “Stain-free technology, reliant on a UV light-driven reaction between halogenated compounds in the acrylamide gel and tryptophan residues on the proteins, provides a more robust and accurate signal than the use of housekeeping proteins,” explains McDonald. “Researchers are often required to demonstrate that expression of housekeeping proteins is unaffected by the experimental conditions, whereas by using the relative total protein signal in each sample lane it is possible to correct easily for variables such as loading error, non-uniform transfer, and loss of protein from the membrane during antibody incubation and wash steps.”
“A limitation of current Western blot documentation systems is that they are not particularly high throughput,” says Kirby, while Isailovic adds that researchers often face a challenge when it comes to reagents. “Many people are still using the ECL they used in graduate school, when so many types of substrate have been developed since then,” she says. “Additionally, while some substrates are designed for film, others are more suitable for digital imagers. Understanding this, as well as knowing when to use chemiluminescence and when to use fluorescence, is something that should be given greater consideration.”
McDonald notes that the imaging system represents the final stage in the Western blot process. “Data quality is dependent on myriad factors, and it is important to record the most accurate representation of the state of the blot, whether it is of poor quality or the cleanest one you ever did,” he says. “You need to trust the result to interpret your experiment correctly.”