As the individual proteins emerge at its mouth, they drop onto a blotting membrane moving steadily across the capillary opening. Proteins travel down a capillary and separate according to size. Kennedy’s laboratory has now swapped the gel electrophoresis step for CE. It didn’t sound like much, but if you’re talking about running many samples over and over again, that simple automation step made life so much easier.”ĬE also requires less sample than gel electrophoresis and has a better resolution of protein size. He adds, “It’s what made the big difference in the Human Genome Project. The advantage of CE is that the sieving matrix for separations can be automatically pumped in and out, because it contains entangled polymers rather than the typical crosslinked polymers of gels, explains Kennedy. The method separates molecules by their size-to-charge ratio inside a narrow electrolyte-filled tube and was the workhorse with which the Human Genome Project was completed (1). Image courtesy of Sha He and Wenying Pan, Jiang Laboratory.Ĭapillary electrophoresis meets protein immunoblottingĬapillary electrophoresis is one way investigators are overhauling the Western blot. Xingyu Jiang and colleagues created a microfluidic device out of a plastic called PDMS to assay a Western blot with 10 different antibodies at once. Furthermore, by increasing the capabilities of Western blotting, those interviewed for the article point out, biologists will be able to formulate more ambitious hypotheses. In fact, a new instrument for Western blotting has appeared on the market that matches that description. “There are very few proteins that are currently used in disease diagnostics of any type, and it’s just because looking for protein biomarkers can be really daunting.”īoth Kennedy and Herr envision an automated system where the researcher can just pop in samples and let the instrument do the grunt work. Herr at the University of California, Berkeley. Today, “there isn’t high-throughput methodology for looking at the protein content and changes in expression and post-translation modifications in large numbers of samples” in an average biology research laboratory, says Amy E. Protein research can be shaken up if Western blotting is automated and made faster with the capacity to analyze many samples at once. Rao says, for example, that her laboratory would love to see an increase in the throughput of Western blotting, because often, “we end up designing our experiments around the number of lanes available on a gel, which is not at all ideal!” I think the time is right” to introduce some innovations to the process, she states. “Traditional Western blotting is tried and trusted, but there are so many limitations to the standard approach. Rajini Rao is a biochemist at Johns Hopkins University who welcomes a redesign. ProteinSimple's Simon uses capillary electrophoresis to fully automate the protein immunoblotting process. “But it’s also true that people spend a lot of time doing Western blots,” says Kennedy. Kennedy acknowledges redesigning Western blotting can be controversial, because it’s valid to question whether there is a need to change something that works. Kennedy at the University of Michigan, Ann Arbor. Maybe it’s time people go back and see if there is anything that can be done” to improve it, says Robert T. But the fundamentals of “this technique have been around for more than 30 years and have hardly changed. Instruments are available to speed up some stages of the process, such as automating the incubation steps with antibodies and blocking buffers or improving visualization and quantitation of the final blot. Its performance is inconsistent and gives rise to variable blotting efficiencies, especially with high-molecular-weight proteins. It can’t process large numbers of samples at once and requires micrograms of proteins, an amount that can be hard to come by for rare or precious samples. However, the multistep technique takes hours and demands technical skill. The power of the method lies in its ability to detect a specific protein in a complex mixture. Since its inception in 1979, Western blotting has been a mainstay in molecular biology and biochemistry laboratories and is used as a confirmatory diagnostic for HIV-AIDS. Photo courtesy of Shi Jin of the Kennedy laboratory The separated proteins emerge from the arrowhead section of the device. Kennedy’s group developed a microfluidic format that separates and blots proteins onto a membrane. Some researchers and at least one company are looking to liberate molecular biologists and biochemists from the manually cumbersome and time-consuming process by rethinking the protein immunoblotting technique. Ever dreamed of the things you could accomplish if you weren’t stuck baby-sitting a Western blot? Help may be on the way.
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