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This Week’s Citation Classic

CC/NUMBER 44

® NOVEMBER 4. 1991 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. 112:195-203. 1981. [Fred Hutchinson Cancer Rcseazch Center. Seattle, WAI A method is described whereby specific antigens can that I might likewise make a solid-phase replica be distinguished in a complex plotein mixture by of a protein gel. After some experimentation, I fractionation in polyacrylamide gels followed by electrophoretic transfer of the protein pattern to ni- found that electrophoresis facilitated the traintrocellulose sheets, detection on the solid phase with for ofproteins from the SOS-gels, that unmodified nitrocellulose worked better 25 than chemiantibody, and visualization by autoradiography. IThe Sd® indicates that this paper has been cited in cally modified paper, and that [‘ llprotein A more than 3,810 publicatiorts.l could bind most types ofantibody-antigen cornplexes3 on blots and was more convenient to prepare than radiolabeling various second antibodies. By blocking nonspecific binding of antiWestern Blotting body and protein A to the nitrocellulose replica W. Neal Burnette of the gel, startlingly dear radiographic images Amgen Inc. of antibody-specific antigens could be obtained. The term “Western” blotting was coined in a Amgen Center Thousand Oaks, CA 91320 distantly remembered discussion with Bob I Nowinski, an allusion to both the geographic In late 1977, I left my postdoc at Albert Ein- location of our laboratory and to Southern and stein College of Medicine, to join Bob Northern blotting. About this same time, the Nowinski’s group at the Hutchinson Cancer protein 4blotting paper of H. Towbin and his colCenter in Seattle. Although my work involved leagues appeared. The general principles of alternative messenger splicing in retroviruses, I their technique and of Western blotting were spent some of my research time contributing to similar; nevertheless, the differences in specific the laboratory’s efforts to map murine leukemia methodology encouraged me to submit a manuvirus structural proteins with monoclonal anti- script to Analytical 8iochemistry. The reviewers bodies. In seeking ways to screen hybridomas were not impressed, objecting especially to the for their epitope specificity, it occurred to me name Western blotting, and the paper was that it should be possible to link principles of the rejected. radioimmunoassay with the resolving power of My move to the Salk Institute obscured my SDS-polyacrylamide gel electrophoresis in order disappointment over failure to publish the techto pinpoint specific antigens in a complex pro- nique. However, the few preprints I had sent to tein mixture (such as a cell extract) with anti- colleagues seemed to have undergone logarithbodies. But it was unclear how I might physically mic Xerox multiplication. I began receiving visualize the in situ interaction of the gel-sepa- phone calls from researchers unable to read the rated proteins with antibody; some of my initial umpteenth photocopied generation of the preattempts to do this were, in retrospect, laugh- print, a sort of technical samizdat that I had to endlessly interpret. With a little prodding, Anaingly naive. To distinguish retrovirus mRNA species, I had lytical Biochemistry eventually agreed to publish been using a solid-phase hybridization proce- the paper, which finally appeared in 1981. Since dure in which gel-fractionated RNA is passively then, Western blotting has become one of the transferred (“blotted”) to activated nitrocellu- most widely employed immunochemical techlose paper and detected with specific radiola- niques, probably a consequence of its relative beled DNA probes i-C.Alwine, et al.’ had hu- simplicity, interdisciplinary applicability, and the morously nicknamed this technique “Northern” visual darity of the results. Perhaps its most blotting in homage to the original developer of important clinical implementation has been as a DNA blotting, EM. Southern.2 It dawned on me confirmatory diagnostic test for AIDS.~

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I. Atwine J C, Kemp Di & Stark G R. Method or detection of specific RNAs in agarose gels by trartsler to diazobenzyloxymethyl-paper and hshridization uith DNA probes. Pnsc. .Vut. .4’ad. St. USA 74:3350-4. 977. (Cited 1.465 times. 2. Southern E M. Detection of specific sequences among DNA fragments separated by gel electrophorevis. J..tfoL Riot, 9s:503-17. 1975. sCited 20.370 times., 3. Langone ii. l’’!lprotein A: a tracer for general ave in immunoavsoy. J. Im,nae,LMrth,sj. 24:169.113. 19711. (Cited 145 times.) 4. Towbin H, Staebelln T & Gordon i. Electrophoretic transfer oi proteins from pois acrs-lamide gels to nitrocellulose sheets: procedore and some applicattons. Proc. Vol. Arod. Sri. USA 76:4350.4. (979. Cited 17.295 times., See also: Towbin H. Citation Clasa,c. Co,’rent C,’ntentu/Lit’e Scienrrt 3I(11c19. 4 March 1988.1 5. Esteban i I. Tal C-C. Kay 3W D. Shihi W-K. Rodner AJ & Alter Hi. lmporsancc of Western blot analysis in predicting infectivity of anti.HTLV-lll/LAV positive blood. Lun,’et 2:1083.6, 9115. Cited 101) t,mcs.i ReceivedOctoberl2. 990

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