January 2012

Revamping the Western blot

 

 western_blot_microfluidic_device 
The microfluidic device developed by Amy E. Herr's group does separation, blotting and antibody binding all in one shot.  Photo courtesy of Amy Herr.

Rao says Jiang’s work is an example of how rethinking Western blotting could change the game. She points out biologists now have to strip and reassay blots if they want to test a sample with multiple antibodies, a process fraught with pitfalls. “If you don’t get a signal, you don’t know if the protein is just not there or you lost it” during the stripping process, she says.

Herr’s group hopes to change the entire Western blot procedure with microfluidics. They recently described an automated system made from polyacrylamide gel that automatically does the electrophoretic separation, transfer and blotting all within its confines (4). Herr says the system requires only 0.01 to 0.5 micrograms of protein. She emphasizes that the system is still a prototype, although her team is collaborating with an industrial partner to commercialize it. Herr wouldn’t reveal the company’s identity except to say it was large.

One of the goals of Herr’s team is to determine precisely the absolute abundance of proteins from rare cells. The group is kicking off a project to characterize proteins in mouse hematopoietic stem cells. “Right now, those [cells] are so sparingly available,” says Herr. “There really isn’t any capability for protein or biochemical characterization of proteins, because levels are so small.”

This is the type of application in which Gary D. Smith, a molecular physiologist at the University of Michigan, Ann Arbor, is very much interested. His laboratory studies glycogen synthase kinase 3’s role in gamete and embryonic development in mammals. Smith explains, “It takes us 300 to 400 oocytes to run one lane on a Western blot. If we do it in triplicate, we’re talking about 1,200 oocytes. Each mouse gives 30 to 40 oocytes.” That’s a lot of dead mice for a Western blot. For this reason, Smith says he is very excited to see the development of miniaturized platforms that could drastically reduce the amount of protein, cells and mice needed for an experiment. Furthermore, both Herr and Smith explain that if microfluidic protein immunoblotting systems let researchers quantify the levels of proteins with different post-translational modifications from just a few cells, and perhaps even single cells, that capability could open new avenues of investigation.

So far, aside from ProteinSimple’s Simon, these methods aren’t commercially available. But the researchers say once they have taken their laboratory prototypes through the development process to become commercial products, they will give biologists the gift of time.

REFERENCES
  1. 1. Zubritsky, E. How analytical chemists saved the Human Genome Project…or at least gave it a helping hand. (2002) Anal. Chem. 74, 23A – 26A.
  2. 2. Anderson, G. J., Cipolla, C. M., and Kennedy, R. T. Western blotting using capillary electrophoresis. (2011) Anal. Chem. 83, 1350 – 1355.
  3. 3. Pan, W., Chen, W., and Jiang, X. Microfluidic Western blot. (2010) Anal. Chem. 82, 3974 – 3976.
  4. 4. He, M. and Herr, A. E. Polyacrylamide gel photopatterninge Enables automated protein immunoblotting in a two-dimensional microdevice. (2010) J. Am. Chem. Soc. 132, 2512 – 2513.

Raj_MukhopadhyayRajendrani Mukhopadhyay (rmukhopadhyay@asbmb.org) is the senior science writer for ASBMB Today and the technical editor for the JBC.

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COMMENTS:

REALLY GREAT IDEA!!!

 

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