March 2012

The men behind Western blotting


ASBMBToday_2012_01_cover

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.

 

 

 

 

 open_channels_burnette   open_channels_stark   open_channels_towbin 
Burnette  Stark Towbin

The Gordon group submitted a description of their method to the Proceedings of the National Academy of Science in June 1979, which appeared in the journal in September (7). The timing of the publications was such that the two groups were unaware of each other’s work until publication.

Stark says his own lab became nitrocellulose converts once appropriate nitrocellulose membranes became commercially available. DBM-paper, which has to be made chemically active prior to immunoblotting, “is not as convenient as just picking up a piece of paper out of a package and blotting directly onto it,” he says. “There is no question that blotting onto the appropriate derivatives of nitrocellulose was the way to go.”

Burnette was unaware of the work done by the two groups as he was developing his approach but saw the Stark and Gordon groups’ papers in print while he was preparing his manuscript. But he felt that his method was different enough to press ahead. He focused on electrophoretically transferring proteins out of SDS-polyacrylamide gels onto nitrocellulose in a more quantitative manner. When Burnette finally got his paper published, he called the approach “Western blotting” in the title and explained the rationale for the name at the end of the introduction (8). For this reason, Burnette is credited for giving protein immunoblotting its nickname (9).

But Stark says his group was calling its method by the same name well before the Burnette paper showed up, although they never used it in their publication. It was completely logical for both Stark and Burnette to come up with the same name: Both were located at research institutions on the West Coast, and the directional joke of blotting was well known by that point (researchers later referred to blotting of post-translational modifications, such as lipids and sugars, as “Eastern blotting”).

The three men have said that they were surprised by the method’s success and longevity. Indeed, apart from changes in detection methods and other tweaks, the method’s principle has persisted unchanged since the 1970s (10).

Towbin is especially amused by how the passage of time has erased the memory of the hard work that went into developing the ubiquitous biochemical tool. “The younger generation of biologists takes the method for granted!” he chuckles.

References
  1. 1) Mukhopadhyay, R. W. Neal Burnette: the man behind the Western blot. ASBMB Today, January 2012:17 – 19.
  2. 2) Renart, J.; Reiser, J.; and Stark, G. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. USA. 1979; 76:3116 – 3120.
  3. 3) Alwine, J.C.; Kemp, D.J.; and Stark, G. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA. 1977;74:5350 – 5354.
  4. 4) Southern, E.M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 1975;98:503 – 517.
  5. 5) Noyes, B.E., and Stark, G. Nucleic acid hybridization using DNA covalently coupled to cellulose. Cell, 1975; Volume 5:301 – 310.
  6. 6) Towbin, H. Origins of protein blotting Biji T. Kurien and R. Hal Scofield (eds.), Methods in Molecular Biology, Protein Blotting and Detection, vol. 536 Humana Press, a part of Springer Science & Business Media, LLC, 2009.
  7. 7) Towbin, H.; Staehelin, T.; and Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 1979;76:4350 – 4354.
  8. 8) Burnette, W.N. “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 1981;112:195 – 203.
  9. 9) Eisenstein, M. A look back: westward expansion. Nature Meth. 2005;2:796.
  10. 10) Mukhopadhyay, R. Revamping the Western blot. ASBMB Today. January 2012:14 – 16.

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


First Name:
Last Name:
Email:
Comment:


Comment on this item:
Rating:
Our comments are moderated. Maximum 1000 characters. We would appreciate it if you signed your name to your comment.


  


COMMENTS:

 

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.

 

0 Comments

Page 1 of 1

found= true1716