More than three decades after its invention, the Western blot remains a crucial tool for investigators who need to reliably identify specific proteins. A host of recently released products use a variety of approaches to improve the reproducibility, sensitivity, quantifiability, and speed of Western blot experiments.
By Anne Harding
Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.
While three different people are considered to have developed protein immunoblotting, just one of them—W. Neal Burnette, then working in the lab of Bob Nowinski at the Hutchinson Cancer Center in Seattle—gets the credit for coming up with the name "Western blotting." The name alludes to Southern blotting (Edwin Southern's 1975 invention of a technique using gel, nitrocellulose, and blotting paper to identify specific DNA sequences in a complex organism), Northern blotting (a similar strategy invented soon afterwards for identifying RNA), and the West Coast location of the Nowinski lab.
Burnette didn't get his technique published until 1981; he recalls that reviewers objected "especially" to the name "Western blotting." Nevertheless, the name stuck, and Western blotting has become one of the most widely used immunochemical techniques.
The first step of a Western blot involves using gel electrophoresis to separate proteins by size, which are transferred to a membrane (usually nitrocellulose or polyvinylidene difluoride) by placing the membrane on top of the gel, adding several sheets of filter paper on top of that, and then placing the entire stack in buffer solution, which draws the proteins up to the membrane using capillary action. This is known as wet or tank blotting.
Two additional techniques, dry and semi-dry blotting, are quicker and less messy than traditional wet blotting, but less effective for high molecular weight proteins. In semi-dry blotting, the membrane and gel are placed between layers of buffer-soaked filter papers, which are in turn sandwiched between an anode and a cathode so an electric current helps drive proteins to the membrane. Dry blotting is the fastest method but has the lowest transfer efficiency.
After blotting is complete, the membrane is placed in a dilute protein solution to block nonspecific protein binding. The membrane is then incubated with the primary antibody, washed, and incubated with a secondary antibody labeled with a probe for signal detection.
Detection, which is usually chemiluminescent or fluorescent, is the final step. In chemiluminescent detection, an enzyme-conjugated secondary antibody produces a light-generating reaction with the detection antigen that can be captured on film or with an imaging device. In fluorescent detection, antibody probes are tagged with fluorophores.
The key advantage of fluorescent detection is that it allows for the simultaneous detection of multiple proteins. Its more consistent signal also means it can be more quantitative than chemiluminescent detection.
Companies making devices, software, and consumables for Western blotting are working to make life a little easier for scientists by automating some—or all—of these steps; adding validation checkpoints so that scientists can check on a blot as it develops; and even reinventing the entire process. Scientists are looking for efficiency, robustness, and strategies that will help them avoid wasting sometimes valuable and costly antibodies.
The blotting, antibody incubations, loading, and wash steps account for 80 percent of the time it takes to do a Western blot, says Michele Hatler, product manager for Western blotting solutions at EMD Millipore.
The Temecula, California-based company's SNAP i.d. 2.0 protein detection system accelerates the process by using a vacuum to drive reagents through the membrane, rather than relying on diffusion alone. This can reduce immunodetection time from four hours to 30 minutes, according to Hatler. "We're really looking at improving the efficiency of workflow in Western blotting," she explains. "We're still following all the traditional steps, we're just adding a vacuum."
The 2.0 version, introduced in September 2012, offers several advantages over its predecessor, according to Hatler. It can be used with midi-sized gels (8.5 cm by 13.5 cm) as well as mini-sized gels (7.5 cm by 8.4 cm), while the older version only ran minis.
"It's very inexpensive and it's very simple, but it really does improve that efficiency and time," Hatler says.
Bio-Rad's Trans-Blot Turbo protein transfer system is a benchtop-size instrument for fast, high-efficiency blotting. Thanks to a new blotting buffer formulation, special filter material, and increased amperage (accommodated by an integrated power supply), the system can complete a blot in as little as three minutes with blot results comparable to tank transfer. Typical semi-dry transfer systems take from 15 to 60 minutes for a complete transfer and often don't provide robust results for transfer of high molecular-weight proteins.
The high failure rates that plague Western blots are particularly frustrating when one has to wait days for a result. "It's a very uncertain technique," says Ryan Short, marketing manager for Western blotting at Bio-Rad. "When we've polled customers we've learned that half of customers report a failure rate of at least 25 percent of their Western blots."
Short adds: "There are very few opportunities to check that the process is going as it's expected. It's anxiety producing. We're introducing the concept of visual checkpoints to add confidence and certainty."
The company's Criterion and Mini-PROTEAN Stain-Free precast gels, used with its ChemiDoc MP gel imaging system, allow investigators to quickly see if their proteins were loaded properly onto the gels, and to verify high-quality protein transfer, providing insight in determining if they should move on to the next step in the process or start over. The ChemiDoc MP system is designed for chemiluminescent and multiplex fluorescent blot imaging and can be operated on a PC or Mac with ImageLab software.
These checkpoints are "really useful," says Aldrin Gomes, an assistant professor at the University of California at Davis who uses the system in his lab. "We can image the stain-free gel without adding an external stain and quickly determine if there's a problem with the samples run on the gel. We can also image the gel after the transfer of proteins to the membrane and if there's a problem at either step we can stop the protocol at this point."
While many scientists still use film for Western blot analysis, there is an increasingly wide range of film-free gel documentation imaging systems available that can be used to image and analyze chemiluminescent blots, fluorescent blots, or both. Companies are competing to make these imagers, and the analysis software accompanying them, as user-friendly as possible. Many offer push-button image capture with a benchtop footprint.
Syngene's PXi, introduced in April 2012, is a compact, one-click system for analyzing both chemiluminescent and fluorescent blots, based on the technology used in the company's G:BOX imaging system and fitted with its GeneSys imaging software.
In October 2012, Thermo Fisher Scientific introduced its myECL Imager, which uses ultraviolet and visible light transillumination, specialized filters, and charge-coupled device (CCD) camera technology to capture and analyze Western blots as well as protein and nucleic acid gels.
The CCD camera is twice as sensitive as X-ray film, according to the company, with 10 times the dynamic range. There is no need to focus or adjust camera settings, because users simply touch one of the optimized presets on the device's touchscreen. Users can set custom exposures—and run up to five different exposures simultaneously—or use preset exposures. The imager has a small instrument footprint, making it suitable for benchtop use.
Aplegen's Omega Lum G gel documentation also features automatic focus and a benchtop footprint, and comes with an integrated tablet computer. UVP's Chemi-Doc-It TS2 imager, released in January 2012, features an integrated computer and touchscreen that allows users to adjust exposure, aperture, zoom, and focus, and also provides an image sample when users touch the Live Preview button.
LI-COR's latest version of its infrared imaging system, the Odyssey CLx, has a dynamic range of more than six-log, compared to more than four-log for its previous version. This increased range eliminates saturation when interpreting Western blots, providing a wider linear range over which to quantitate data.
"Not only will researchers not get saturation on their blots, but they can put multiple blots onto the imager and never have to worry about adjusting the blots to different settings," explains Jeff Harford, senior product marketing manager at the Lincoln, Nebraska company.
Rehana Leak is an assistant professor at Duquesne University in Pittsburgh who uses the LI-COR's Odyssey CLx in her research on how cells adapt to low levels of stress. "The advantage of the Odyssey [imager] is the 16-bit imager and use of two infrared wavelengths, 700 and 800 nm, to visualize two stains at once," Leak says. "This means that the phosphorylated and total forms of proteins can be detected simultaneously, for example." Two protein isoforms can be difficult to distinguish simultaneously by other methods because their masses are so similar.
Leak points out that the Odyssey imager can visualize 216 shades of infrared signal, compared with the 150 shades of gray visualized on X-ray film. "You're going from 150 to over 65,000 shades of infrared, so you have much higher resolution with this method," she adds. "It's unusually quantitative."
In December 2012, LI-COR announced a new digital imaging system, the C-DiGit System, which can be used with chemiluminescent blots and will be released some time in 2013. "Just as digital cameras replaced film-based cameras in the last few years, we see the C-DiGit System as a legitimate replacement for film-based chemiluminescence," says Jon Anderson, a senior scientist at LI-COR. "The technology provides all the sensitivity and image quality that film users are accustomed to, while providing significant improvements in digital image quality."
Traditional software for analyzing Western blots requires users to export data from the image to a spreadsheet, where further normalization calculations and analysis are completed manually. BioRad's Image Lab 4.1 software, released in September 2012, provides built-in, automated normalization to either a housekeeping protein or to a total lane protein.
"It's one of the best I've seen on the market now," says Gomes, who studies the role of proteasomes and troponins in cardiac and skeletal muscle tissues. "Best of all, it's free." Using Image Lab 4.1 is easier than other image analysis software his lab previously used, adds Gomes, and just about anyone can handle it. "It really has enhanced how we quantify the proteins."
LI-COR has also introduced new imaging software, Image Studio, for use with its Odyssey systems. Users choose a detection channel, and then push a button to acquire an image. The new version of the software allows for analysis in millimeter increments, rather than centimeter increments, and can perform completely automated analysis as well as manual analysis.
Instead of tweaking the traditional Western blot, ProteinSimple's Simple Western has reinvented it, using capillary electrophoresis to fully automate the process. In 2011, the company introduced Simon, an instrument designed to completely replace the Western blot. Simon performs size-based separation, immunoprobing, washing, chemiluminescent detection, and quantitative analysis. Scientists simply load a sample, walk away, and come back later to fully analyzed data. Simon even sends out a tweet to let you know your results are ready.
Next came Sally, in early 2012, which allows users to perform 96 Simple Westerns in one experiment; the latest device is Peggy, introduced in September 2012, which can analyze 96 samples at once and separates proteins by size or charge. The Simple Western overcomes the drawbacks of the manual aspects of the traditional Western, namely lack of reproducibility or quantitative results. In addition, multiplexed detection in a traditional Western has been a challenge. Investigators can now use fluorescence to look at two or three proteins within a sample, and transfer devices that run up to eight mini- or four midi-gels at the same time, but that's as far as traditional Western blotting can go. Simple Western can multiplex proteins in every capillary, essentially eliminating this limitation of the traditional Western.
For Alice Fan, an instructor in oncology at Stanford University School of Medicine, the most exciting thing about the Simple Western technology is its ability to analyze nanoliter-sized samples.
Fan has been banking patient tissue samples for more than a decade, waiting for a tool that will let her analyze the proteomics of tumor cells without using up the entire sample. "I've been looking for something for years that will let me be able to do a Western blot with very small amounts of tissue," she explains.
In addition to helping her to conserve precious banked tissue, Sally has made it feasible for Fan to take several samples from an individual patient over time using fine-needle aspiration, rather than doing a full-fledged biopsy. This, Fan hopes, will let her watch how a patient's tumor is responding to a drug in real-time. "This really helped my research in terms of how to profile different tumors. It's a huge leap forward."
Richard Rustandi, chair of the vaccine biochemistry group at Merck & Co., didn't have high hopes when he purchased a Simon instrument at the end of 2011. But, he says, he's been pleasantly surprised. "We liked it so much we actually purchased a second one a few months later."
According to Rustandi, while Simon is not perfect, it can do true quantitation. And while manual Westerns typically have 35 percent to 45 percent variability, he adds, he and his lab have pegged Simon's variability at less than 10 percent.
As Western blotting technologies continue to evolve, researchers are finally able to start spending a little—or a lot—less time babysitting their experiments. "Their time is important," says Peter Fung, the product manager for Simple Western. "And the more efficient you can be with your time, the better research you can do."
Merck & Co
Stanford University School of Medicine
Thermo Fisher Scientific
University of California at Davis
Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. Inclusion of companies in his article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.
Anne Harding is a freelance science writer based near New York City.
|This article was published as a special advertising feature in the 1 March 2013 issue of Science magazine.|
New transilluminators with high-performance blue light LEDs are available for gel documentation purposes. Blue light transilluminators are an interesting alternative to ultraviolet (UV) tables as there is no risk of sample damage during illumination, which is important for processing samples following gel documentation. The transilluminators also benefit users, as there is no risk of UV exposure. Blue light excitation can be used with fluorescent dyes for nucleic acid or for protein stains that have an excitation wavelength around 470 nm. Examples of compatible stains are: SYBR Green, GelGreenTM, SYBR Safe, SYBR Gold, and SYPRO Ruby. The instrument's amber lid allows direct viewing of the fluorescent samples, with no need for separate glasses. The new blue light illuminators are available in two versions: the BLstar 9, a handy solution for mini gels, and the BLstar 16, for gels up to size of 16 cm x 20 cm.
The Image Lab 4.1 software is designed for use with the ChemiDoc MP imaging system. The new software automates multiplex analysis, providing a faster, more accurate alternative for quantifying protein expression levels via Western blotting. Proving that quantitative differences of a target protein are a result of biological differences and not experimental variability (such as unequal protein loading) is critical when conducting Western blotting experiments. Typically, scientists comparing gene expression levels use a housekeeping protein (HKP) as a loading control with the assumption that HKP expression does not change based on treatment type or sample origin. However, total protein normalization has been shown to be more stable than HKP normalization regardless of conditions or sample type and saves time by eliminating the need for multiple probing steps or optimizing a multiplex blot. With Image Lab 4.1 software, researchers can deliver normalized intensities automatically.
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The WestVision Peroxidase Polymer Anti-Rabbit IgG and Peroxidase Polymer Anti-Mouse IgG are designed to detect primary antibodies of mouse or rabbit origin in Western blots. The WestVision conjugates are based on a new method of polymerizing enzymes and attaching these polymers to antibodies that can allow a higher density of enzymes to access a target with lower interference. With a chromogenic substrate, the reagents can provide enough working solution to typically detect 20 to 200 blots (10 cm x 10 cm). With a chemiluminescent substrate, dependent on the dilution employed, 800 to 8,000 blots can be detected.
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A new line of chemiluminescent Western blot kits provide the most sensitive detection solutions yet. Forty-five percent of researchers recently surveyed cited a lack of sensitivity and time to results in Western blots as significant challenges. The search for increased sensitivity can amplify background, requiring lengthy optimization to manage signal:noise contrast. SignaLOCK ChemiWestern kits solve these challenges. Each kit is composed of a chemiluminescent substrate, a non-protein blocker, and unique wash solution. All kits offer comprehensive protocols and can be used with both nitrocellulose and PVDF members. Whether using film or an imager, there is an optimized kit that will enable researchers to see more signal with less background in their Western blots.
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Low protein binding microtubes are now offered in both 0.5 mL and 1.5 mL volumes. These tubes are specially designed to meet the requirements of protein analytics while maximizing protein recovery rates. The microtubes are manufactured from a proprietary polypropylene with low protein binding properties and do not feature any potentially contaminating surface coating like silicone. The low protein binding microtubes feature an attached flat SafeSeal cap with a tooth-like cam that locks over the tube rim, increasing retaining strength and providing a reliable, secure closure. Tubes can be centrifuged to 20,000 x g, and are polymerase chain reaction (PCR) Performance Tested, and therefore certified to be free of human DNA, DNase, RNase, and PCR inhibitors.
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