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.
Western blotting—widely relied upon for identifying specific proteins—is not known for its speed or throughput. It is, however, prized for its accuracy and reliability in a wide range of life-science applications. “People usually run Western blots to confirm a finding,” says Russ Yukhananov, CEO of Precision Biosystems. “It is not an exploratory method, so the quality of the results is critical.” With many other assays receiving high-throughput “makeovers,” it is tempting to entertain the notion of high-throughput Western blotting. Here are some tools to help boost Western blotting throughput—without sacrificing accuracy.
Increasing efficiency
Making Westerns more efficient means saving time, which itself boosts throughput. EMD Millipore’s SNAP i.d.® 2.0 Protein Detection System accomplishes this by using a vacuum to draw buffers across the blotting membrane, increasing “the efficiency of mass transfer by convective transport rather than simple diffusion,” says Timothy Nadler, senior research and development manager at EMD Millipore. The SNAP i.d. system uses blotting membranes, encompassing the Western probing steps (such as blocking, antibody incubations and washing) that occur after transferring the proteins from gel to membrane. For an efficient transfer system, Bio-Rad Laboratories’ Trans-Blot® Turbo™ Transfer System rapidly transfers proteins from gel to blotting membrane in about three minutes and also integrates into the company’s V3 Western Workflow™ for protein separation, detection and analysis.
Bio-Rad’s V3 Workflow introduces quality-control checkpoints into the blotting protocol. “Western blotting is like a black box to the experimenter,” says Kris Simonyi, product manager at Bio-Rad. “Typically, one has to wait until the last minute of the long process (up to two days) to find out that something went wrong.” At each checkpoint, the researcher can verify that the blot is proceeding properly—checking for good separation immediately after gel electrophoresis, for example, or for good transfer.
Addressing efficiency in workflow, GE Healthcare Life Sciences recently released the Amersham WB System, which monitors every stage of the Western blotting process, including electrophoresis, transfer, fluorescent probing and scanning to provide quantitative protein analysis. Thereby, minimizing sources of variability and generating more consistent, quantitative results.
In principle, multiplexing—testing for multiple proteins simultaneously—can also increase throughput. Some labs accomplish this by using up to three target antibodies at once. But it can be a feat of optimization, according to Simonyi: “Very few labs do multiplex fluorescent blots due to many optimization challenges relating to antibody quality, cross-reactivity, background issues and insufficient fluorescence sensitivity needed for low-expression or weak antibodies.” Nadler states that instead of fluorescent detection, chemiluminescent or chromogenic reagents can be used for single targets or targets with sufficiently different molecular weights.
For multiplexing fluorescence antibodies, says John Lyssand, field applications scientist at LI-COR Biosciences, it is important to consider “how sensitive will my detection tag be, and is there sufficient spectral separation between my tags to avoid cross-talk?” LI-COR uses fluorescent tags in the near-infrared wavelengths to reduce background signal.
Antibody quality is also key for boosting throughput, because antibodies that lack the required specificity or sensitivity can yield equivocal results. “Many Western blot experiments fail because of poor antibody quality,” says Ning Liu, senior product manager at Bio-Rad, who believes it is important to set high standards for antibody validation. “Don’t use antibodies validated with overexpressed proteins,” he says. “We only rely on endogenous protein levels for testing and check 12 different cell lines or tissues.” In 2015, Bio-Rad is planning to release a PrecisionAb line of antibodies validated for Western blotting.
Running more samples in parallel
Another way to increase throughput without sacrificing quality is to use multiple large blots in parallel. Increasing blot size—for example, by using midi- instead of minigels—can roughly double the number of samples running simultaneously. Also, many gel systems can run more than one gel in the electrophoresis tank or blotting apparatus. For example, Bio-Rad’s Trans-Blot Turbo can run up to four minigels or two midigels simultaneously. Sometimes this alone is enough of a throughput bump, especially for smaller labs, notes Kevin Lowitz, senior product manager in protein biology at Thermo Fisher Scientific. “Compared to semi or fully automated integrated systems, it can be more cost-effective to have multiple personal benchtop devices set up to increase throughput,” he says.
Similarly, probing multiple blots simultaneously can speed throughput. EMD Millipore’s Multiblot Frame can increase the SNAP i.d. system’s maximum capacity to four blots every 30 minutes—up to 64 blots in eight hours. “Additionally, the blot frames can be removed from the SNAP i.d. 2.0 Protein Detection System manifold during antibody incubations, so throughput could be increased beyond 64 blots per system,” says Nadler. LI-COR Biosciences’ Odyssey® CLx system can image up to nine minigel-sized Western blots simultaneously. LI-COR’s MPX™ Multiplexing blotting device also “allows researchers to probe a single sample with up to 48 antibodies at once on our Odyssey systems,” says Lyssand.
Precision Biosystems’ BlotCycler™ can process up to 12 blots simultaneously. The company’s patented fluidic technology does the blocking, antibody incubation and washing automatically. “The system can handle up to 150 protein samples in minigel format or up to 220 protein samples in midigel format,” says Yukhananov. Researchers can “continuously run multiple blots with multiple antibodies,” he says. “In 10 working days, they can process almost 2,000 protein samples.” He stresses, however, that Precision Biosystems places its emphasis on the quality of the results, not the number of samples processed.
Nontraditional formats
Nontraditional takes on Western blotting have given rise to protein assays with higher throughput than traditional Westerns, often using multiwell plates, assay strips or even capillary tubes. For example, Tecan’s automated ProfiBlot™ 48 Western blot analyzer uses assay strips to process up to 48 samples at once. ProteinSimple’s Simple Western™ systems automate runs of up to 96 samples simultaneously. Instead of traditional gel electrophoresis, the Simple Western utilizes capillary electrophoresis to separate proteins, followed by a chemiluminescent immunodetection in the capillary and full analysis of the results upon completion of the experiment
LI-COR’s plate-based In-Cell Western™ Assay uses multiwell plates to save time, because the initial steps of lysing cells, running gels and transferring to a membrane are unnecessary. A standard 384-well plate yields 384 datapoints in one run. “To obtain a similar amount of data from [traditional] Western blotting, a researcher would need to run nearly 43 10-well gels, assuming one lane is used for a molecular-weight marker,” says Lyssand.
The throughput of the In-Cell Western can be further extended by using LI-COR’s Odyssey CLx, which uses up to six multiwell plates simultaneously. At maximum, an Odyssey CLx running six plates of 1,536 wells each results in “9,216 samples that can be analyzed at once,” says Lyssand. The similar Odyssey Sa imaging system comes with an optional 50-plate capacity plate stacker to expand automation capabilities.
But some nontraditional formats are best used after your traditional Western blots are working well. “Once reagents and detection systems have been fully validated, it may be possible to switch from a Western blotting format to a dot blot configuration,” suggests Nadler. “Filter plates with blotting membrane, like the MultiScreen®-IP Filter Plate, can be used to automate protein capture, probing and detection in a 96-well format.”
In considering nontraditional formats, Lyssand says they’re best used in particular circumstances. If researchers are studying multiple proteins differing in molecular weights, such as with degradation or cleavage products, or if their primary antibody is nonspecific, then traditional Western formats are the best choice. But nontraditional formats may be suitable “if a researcher is interested in a single target, and if [the] primary antibody is specific for its target.”
Ways to boost Western blotting throughput are growing—but slowly and carefully, which is a good thing. “While increasing throughput is valued by some researchers, the ability to detect specific proteins in a complex sample with a broad range of affinity reagents and with minimal optimization and better reproducibility continue to be critical factors when evaluating new technologies for Western blotting,” says Lowitz. Because, as anyone who relies on Western blotting will tell you, its strengths—sensitivity and accuracy—should never be sacrificed for throughput.
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