Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
In the old days, all that was required for protein gel documentation was a Polaroid camera. Today, film has given way to bits and bytes, producing digital images that are blots are more quantitative and higher resolution than film, not to mention archivable and searchable.
Given the many options, selecting a system for protein and Western blot documentation can be overwhelming. For example, Western blots can be read using chemiluminescent, fluorescent or colorimetric assays. There are also different kinds of light sources, imaging modes and applications. Which is right for you? Here's what you need to know.
Key considerations
As with any instrumentation purchase, the first important consideration is application: What do you need your imager to do? Chemiluminescent or fluorescent? One fluorescent color or two? “Consider which applications they would like to image, and also the viewing area and sample size they require, as this will help to narrow down their options,” says Lindsey Kirby, applications specialist at Syngene, which is part of Synoptics.
Also, will your lab will use the system for blots exclusively, or also for gels? (If so, do you need to image protein acrylamide gels only, or also DNA agarose?). “For the combined use, the most convenient solution is a system with a zoom lens,” says Stefanie Navabi, product manager in life science at Biometra (part of Analytik Jena). “This allows the simple and flexible work with smaller and larger gels and blots.” On the other hand, researchers who image only Western blots may be better off with a camera lens that has a fixed focal length. “These customers usually wish [for] great sensitivity,” says Navabi. “A lens with fixed focal length is much more light-sensitive than a motorized zoom lens.”
Researchers who need to maximize sensitivity (for example, to image a faint band of a low-abundance protein above background noise or to resolve two closely spaced bands) should consider camera types. “Look for a cooled CCD camera in an imaging system, because it delivers increased sensitivity by decreasing ‘dark’ or thermal noise,” says Priya Rangaraj, market development manager at Thermo Fisher Scientific. Other camera characteristics that contribute to image quality include pixel size, lens and aperture (aka F-stop, whose value is inversely proportional to the degree of lens opening). “A larger diameter aperture ... allows more light to reach the image sensor,” explains Rangaraj. “Sensors equipped with larger pixels collect more light, which translates into better sensitivity and image quality”—less noise, in other words.
The camera resolution is the most important factor in choosing an imaging system, says Patrick Smith, inside sales representative in imaging at ProteinSimple. “The higher the resolution, the easier it is for you to see the separation of two bands that are close to each other,” he says, adding that another important factor is dynamic range. “Dynamic range is the number of shades of grey you will see, represented by the bit-depth,” he explains. “Higher bit depths allow for better dynamic range.”
Another important consideration is reproducibility. “Does signal double if I double the amount of protein?” says Jeffrey Harford, product marketing manager at LI-COR Biosciences. And what about the variance of the data, expressed as a CV%. “Can I image the same blot repeatedly and get the same result [indicating a] low CV%?” he says. “What if the blot is moved around—do I still get a low CV%?”
Finally, consider user-friendliness. A system that’s easy to navigate will help researchers get better data, faster. Take the system for a test-drive, suggests Lisa Isailovic, director of marketing at Azure Biosystems. “You need to have hands-on experience with the system to answer the questions Is it easy to use? Does it image my samples? Does it give me the results I expect?”
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Be sure to try to the system’s software, too. “In many cases, free trial software is available,” says Rangaraj. “Try it out and be the judge of how user-friendly it is.” Another indication of imaging system user-friendliness is whether it is plug-and-play or requires user training at the time of installation.
A system sampler
Analytik Jena’s ChemStudio chemiluminescent systems use cooled CCD cameras to reduce background noise and enhance sensitivity. Similarly, for low-level signals, they offer sealed darkrooms to block out light during long acquisition times. The company’s systems use transillumination with UV, blue or white light, and they also can read fluorescent and colorimetric blots. System prices vary, says Navabi, according to the features you need. “Depending on desired sensitivity, there are different cameras and lenses available—the higher the sensitivity, the higher the price.” The company’s Western-blot imaging systems range in price from approximately €12,000 to €40,000.
Azure Biosystems offers the cSeries line of Western-blot imagers, which use visible to near-IR light for fluorescent blots. They also can detect chemiluminescent blots. The company’s models range in price from $8,000 to $49,000, and can also be used for agarose gel imaging and protein gels.
Bio-Rad Laboratories offers three tiers of Western-blot imagers, starting with the ChemiDoc™ XRS+ system. The middle-tier model is the basic ChemiDoc MP system, and the highest-tier model is the fully loaded ChemiDoc MP. The systems include a cooled CCD camera for detecting faint signals. Depending on which optional features are added, a ChemiDoc system can detect chemiluminescent and colorimetric signals and up to three colors for fluorescent multiplexing. The imaging systems are also compatible with Bio-Rad’s Stain-Free technology, which enables users to check the gel and blot for protein profile and quantity at any point for quality control. Bio-Rad’s imagers range from $20,000 to about $45,000, depending on the model.
GE Healthcare offers two types of Western-blot imagers, with different capabilities. The lower-end ImageQuant LAS 500 (costing less than $20,000) is designed to read chemiluminescent signals. GE’s higher-end Amersham Imager 600 (costing $25,000 to $40,000) also can multiplex up to three fluorescent signals (using blue, green and red filters), as well as image samples in the visual spectrum, according to product manager Joe Hirano.
LI-COR offers a range of imagers that differ in their applications. The C-DiGit® Blot Scanner reads chemiluminescent Western blots, and the Odyssey® Fc Imaging System reads chemiluminescent and infrared blots in addition to DNA agarose gels. The Odyssey CLx and Odyssey Sa Imaging Systems use infrared lasers for illumination for infrared Western blots, DNA agarose gels and other protein applications.
ProteinSimple (recently acquired by Bio-Techne) offers a range of Western-blot imagers in its FluorChem™ line, with different models having different functionalities. The FluorChem R, for instance, can image infrared fluorescence signals, while models R, M and Q have three-color fluorescence capability. The FluorChem E system is dedicated to chemiluminescent Westerns. The FluorChem R, M and E systems are designed to be especially easy to use—no computer connection is required as the unit contains an internal computer, and the systems' progress can be monitored remotely with any browser-enabled device using ProteinSimple's Digital Darkroom software.
Syngene makes two imaging system lines: the G:BOX Chemi and the smaller, more compact PXi. According to Kirby, the systems can read fluorescent and chemiluminescent blots, bioluminescence and multiplexed samples. The G:BOX Chemi systems range from $13,622 to $24,514; the PXi systems range from $15,396 to $21,778.
Thermo Fisher Scientific's myECL Imager system can read chemiluminescent, fluorescent and colorimetric blots and is able to switch among modes for different uses. The system uses UV and visible transillumination. In addition to detecting protein on Western blots, the myECL Imager can be configured to detect DNA on agarose gels.
UVP’s BioSpectrum® Imaging System uses UV, blue and visible light. The company also offers the BioLite™ MultiSpectral Light source, which excites samples using red, blue, green and near-infrared light. According to UVP product manager Mike Capps, list prices for UVP’s systems range “from the mid $3,000s up to the mid $30,000s for fully featured gel and chemiluminescence imaging and analysis packages.”
With the many Western-blotting systems available today, you're sure to find one that will fulfill your experimental and technical needs. But don’t forget ease of use, says Ning Liu, product manager for Western blotting in the Laboratory Separation Division at Bio-Rad. “[Researchers] don’t really want to spend too much time to figure out how to use a complicated system,” says Liu, “because scientists are not technologists.” With the wide selection of imagers available today, you can spend more time interpreting your data—and less time struggling to generate it.