January 2012

Revamping the Western blot

Researchers are pursuing efficiencies, automation and higher throughput



 

 western_blot_penny 
Robert T. Kennedy’s group developed a microfluidic format that separates and blots proteins onto a membrane. The separated proteins emerge from the arrowhead section of the device. Penny shown for scale. Photo courtesy of Shi Jin of the Kennedy laboratory.

Ever dreamed of the things you could accomplish if you weren’t stuck baby-sitting a Western blot? Help may be on the way. 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.

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. The power of the method lies in its ability to detect a specific protein in a complex mixture. However, the multistep technique takes hours and demands technical skill. 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. Its performance is inconsistent and gives rise to variable blotting efficiencies, especially with high-molecular-weight proteins.

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. But the fundamentals of “this technique have been around for more than 30 years and have hardly changed. Maybe it’s time people go back and see if there is anything that can be done” to improve it, says Robert T. Kennedy at the University of Michigan, Ann Arbor.

Kennedy acknowledges redesigning Western blotting can be controversial, because it’s valid to question whether there is a need to change something that works. “But it’s also true that people spend a lot of time doing Western blots,” says Kennedy.

western_blot_proteinsimple 
ProteinSimple's Simon uses capillary electrophoresis to fully automate the protein immunoblotting process.  Photo courtesy of ProteinSimple.

Rajini Rao is a biochemist at Johns Hopkins University who welcomes a redesign. “Traditional Western blotting is tried and trusted, but there are so many limitations to the standard approach. I think the time is right” to introduce some innovations to the process, she states. 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!”

Protein research can be shaken up if Western blotting is automated and made faster with the capacity to analyze many samples at once. 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. Herr at the University of California, Berkeley. “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.”

Both Kennedy and Herr envision an automated system where the researcher can just pop in samples and let the instrument do the grunt work. In fact, a new instrument for Western blotting has appeared on the market that matches that description. Furthermore, by increasing the capabilities of Western blotting, those interviewed for the article point out, biologists will be able to formulate more ambitious hypotheses.

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COMMENTS:

REALLY GREAT IDEA!!!

 

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