March 2012

The men behind Western blotting


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In the January 2012 issue of ASBMB Today, I profiled American Society for Biochemistry and Molecular Biology member W. Neal Burnette, describing him as the man who developed Western blotting (1). In doing so, I might have inadvertently given some readers the impression that Burnette was the only person who worked on protein immunoblotting. But it’s critical to note that two other groups had made important contributions to the technique in the late 1970s and published their findings before Burnette was able to do so.

George Stark, now at the Cleveland Clinic but then at Stanford University, and his colleagues published the first paper that described the transfer of proteins by capillary action from a polyacrylamide/agarose gel with or without the denaturing agent SDS onto a special membrane called diazobenzyloxymethyl-paper (2). The membrane is called DBM-paper for short. Stark’s group was already famous for developing the RNA blotting technique known as “Northern blotting” (3). The name was a joke based on the DNA blotting technique called “Southern blotting,” which was named after its inventor, Edwin Southern at Oxford University (4). It was for Northern blotting, Stark explains, that his laboratory became experts in making DBM-paper. In 1975, Stark’s group had described how to make chemically reactive cellulose that would covalently bind to DNA and RNA (5). This powdered cellulose could be used to isolate complementary nucleic acids by hybridization. This chemistry was the basis for Northern blotting, but Stark’s group used paper rather than powdered cellulose for the method.

“Following on from that, we soon realized that there was a problem in detecting specific proteins,” says Stark. They used the same chemistry as for Northern blotting, because they realized DBM-paper reacted with both nucleic acids and proteins. When the proteins transferred out of the gel, they covalently bound to the DBM-paper. “The surprise bit was that the immobilized protein reacted so nicely and specifically with antibodies,” notes Stark. They submitted a description of their method to the Proceedings of the National Academy of Sciences in April 1979, and the paper appeared in July.

Meanwhile, over in Europe, Harry Towbin was a postdoctoral fellow in the laboratory of Julian Gordon at the Friedrich Miescher Institute for Biomedical Research in Switzerland. Like Burnette, Towbin was dealing with an analysis problem in his research project, which focused on making antibodies against ribosomes for structure-function studies (6). Towbin’s problem was much like Burnette’s: Both were trying to figure out the specificity of antibodies against proteins in complex macromolecular structures but realized they didn’t have an easy and reliable biochemical tool for the analysis.

Unaware of the work being done by Stark’s group, Towbin and Gordon, along with Theophil Staehelin at Roche, began to work out a method that would allow them to establish which antibody bound to which component of the ribosomal complex. By that point, DNA and RNA blotting methods were popular, so the idea of transferring proteins out of a gel and onto a membrane seemed natural. “It was in the air!” says Towbin, who is now set to retire from the Swiss Federal Institute of Technology Zurich in April.

He says they knew that proteins, but not RNA, bound to nitrocellulose, so they separated ribosomal proteins on a polyacrylamide gel with urea as a denaturing agent and then electrophoretically transferred them onto nitrocellulose. The proteins noncovalently clung to the nitrocellulose, but RNA didn’t; this feature nicely eliminated the nucleic acid from the proteins. Although they primarily focused on gels with urea as the denaturing agent, Towbin says they also got their approach to work with SDS.

 

 

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Note to the author: when referring to RNA or DNA blotting, it should not be called immunoblotting since doesn't rely on the use of antibodies to detect molecules as in western blotting. Editor's note: Thank you. The article has been corrected.

 

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