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Analytical Chemistry

Capillary Electrophoresis Goes Western

Protein Separation: A new method demands less time and sample than does conventional Western blotting

by Rajendrani Mukhopadhyay
February 2, 2011

Biochemists may soon have more time on their hands. Researchers in Michigan have speeded up Western blotting, a biochemistry workhorse for protein separations, by coupling it to an analytical chemistry mainstay, capillary electrophoresis (CE) (Anal. Chem., DOI 10.1021/ac102671n). The new technique produces data in a quarter of the time, and with a fraction of the sample, that Western blots normally use.

Western blotting detects a specific protein in a mixture. The technique takes three steps that begin with electrophoresis separating the individual proteins into bands on a gel that look like rungs on a ladder. In the next step, a voltage transfers the proteins from the gel onto a membrane. Researchers then treat the gel with specific antibodies in a final step to produce a blot that shows which rung corresponds to the protein of interest. The technique takes four hours or longer. Although Western blotting is a ubiquitous tool, very few people have gone back to the drawing board to see how to improve it, says analytical chemist Robert Kennedy, of the University of Michigan, Ann Arbor.

Kennedy's laboratory specializes in CE, which separates molecules inside a small electrolyte-filled capillary tube according to their size-to-charge ratio. Compared to gel electrophoresis, CE uses less sample and applies higher electric fields. As a result, it goes faster and achieves better resolution, explains Kennedy. CE also lends itself well to automation, he adds.

So graduate students Gwendolyn Anderson and Cynthia Cipolla in Kennedy's laboratory swapped gel electrophoresis for CE to separate proteins. Proteins migrated through a capillary, and as the individual proteins emerged at its mouth, they dropped onto a blotting membrane moving steadily across the capillary opening. "You are separating and blotting at the same time," says Kennedy. The result is eliminating the labor-intensive gel-to-membrane transfer.

The investigators tested classic protein standards including carbonic anhydrase and lysozyme to show that an entire Western blot inside a capillary took only about an hour. They could detect low picograms of material, about the same sensitivity as conventional Western blotting. The CE-based method used only a few nanoliters of sample, whereas the conventional Western blot sucks up microliters.

Kennedy emphasizes that the current work is a preliminary step. His team is working to automate a system with multiple capillaries in an array, to enable separating proteins from many samples at once. He also intends to use microfluidics to further shrink the system so that it has a smaller footprint.

Cutting out the gel-to-membrane transfer step is the method's best attribute, says biochemist Rajini Rao at Johns Hopkins University, whose laboratory runs Western blots daily. It means "one less piece of equipment, fewer buffers to make, and less chance for mistakes." However, she says that to appeal to biologists, the setup has to be optimized to be easy to operate.

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