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Program in Cell Dynamics, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
1Correspondence: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 377 Plantation St., Worcester, MA 01605, USA. E-mail: thoru.pederson{at}umassmed.edu
ABSTRACT
The zonal separation of proteins on the basis of net charge was initially conducted on paper, then in columns of sucrose and later in gels of starch and polyacrylamide, with appropriate electric fields. Then, in 1964, a graduate student at MIT discovered the power of sodium dodecyl sulfate (SDS) to dissociate the envelope proteins of Escherichia coli and to dramatically enhance their electrophoretic resolution when the detergent was included in the gel. While this Ph.D. thesis work continued, a group at the Albert Einstein College of Medicine published in 1965 the use of SDS to disrupt poliovirus particles and to resolve the proteins in gels containing SDS. This group soon followed with a publication (1966) on the application of this new method to the study of immunoglobulin heavy and light chain synthesis. Because of concurrent advances in gel filtration and other methods of protein separation, SDS gel electrophoresis had its greatest impact not in biochemistry but in cell biology and virology. Ingenious devices were soon introduced that facilitated the application of this method to radioactive protein mixtures, followed by the introduction of slab gels for the simultaneous resolution of multiple samples in parallel lanes in a single run. As we today routinely perform "SDS PAGE" (as the method become known, to the great irritation of journal copyeditors and nomenclature committees at the time), it is fitting to pause—four decades later, and remember the pioneers who made SDS gel electrophoresis a reality, a true milestone that caught on almost overnight.—Pederson, T. Turning a PAGE: the overnight sensation of SDS-polycrylamide gel electrophoresis.
WHEN I WAS AN UNDERGRADUATE at Syracuse University I dabbled in English (but with no hope because professors were still buzzing about Joyce Carol Oates who had blown them away a few years before), then Latin (pleasurable but with no job future), chemistry (initial enthusiasm diminished by a lab partner’s repeated use of n-amyl mercaptan in nauseating amounts and a bromination reaction of mine that got out of hand), I came—at last—to biology. But one problem: there was no biochemistry course. For that we went over to the State University of New York School of Forestry. (It was located behind the Syracuse University football stadium, where Jim Brown had run over absolutely everything in his way a few years before.) The Forestry School biochemistry professor was Ernest Sondheimer, an expert on terpenes and related natural products in trees. He bore a striking resemblance (and manner) to the actor Peter Lorre, lecturing with a cigarette dangling from a corner of his mouth—imagine this today! Professor Sondheimer always threw an extra credit question onto the exam, baiting us to try to move up from the 75–85 scores he typically handed out in his tough grading métier. One such question that I particularly recall was: "What is the best definition of life?" In my blue book (for readers under 55, these were the sheaves of paper, stapled inside a blue cover, on which we wrote exam answers in the pre-Cenozoic era). I wrote a few lines on the subject of self-replication, an answer so primitive as to perhaps match up with the pre-biotic timescale of the question. Dr. Sondheimer wrote "Nice try" on my graded exam but the answer he wanted was "anything that can separate stereoisomers of a substance." After class I asked him if by this he meant a primordial concretion of clay and carbon chains that had, by random events, become oriented at nonrotational bonds so as to somehow be tuned to differentially handling racemic mixtures of things floating by. He replied, "Yes—that’s the idea." This memory stuck. When I read, 20 years later, about Louis Pasteur pushing crystals of D- vs. L-tartaric acid to one or the other side with forceps under his microscope (1)
, my professor’s definition took on new meaning. Today there are drug companies that have proprietary methods for pulling out the bioactive stereoisomer from a synthesized racemic mixture when the commercially viable synthetic route is not stereospecific. The materials used in these methods are not alive, but the people who invented them and use them are. The fact that I am still thinking about my professor’s question doesn’t mean I know the definition of life. What it means is that I had a good biochemistry teacher.
One of Professor Sondheimer’s other extra credit questions was: "What is the most important concept in biochemistry so far?" Although this was a decade past the double helix, his answer was: "The discovery that enzymes are proteins." (I had woefully written "paper chromatography" which, although wrong, again got me one of his "nice try" responses.) He argued that the concept that enzymes are proteins was the seminal event that linked physiology to chemistry and thus gave the term "biochemistry" its true credentials, as well as accelerating the development of improved methods to purify and resolve proteins. He was absolutely right, of course.
The separation of proteins from complex mixtures represented one of the major challenges in biochemistry in the early 20th century, once the general foundations of protein composition and chemistry had been established. In Stockholm, Arne Tiselius pioneered the electrochemical view of proteins both as a chemical concept and as a property that could, as he so presciently saw, be exploited in various ways, an insight for which he received the 1948 Nobel Prize in Chemistry. Subsequently, electrophoresis of proteins was conducted in columns or sheets of polymers such as starch or polyacrylamide. In due course, this led to the discovery that some enzymes are present in certain cells or organisms in multiple forms sharing catalytic properties but differing in subunit composition and net charge, soon termed "isozymes." A famous story has a pioneer in this field, Nathan Kaplan, serving as an expert witness in a lawsuit in which a Boston fish market (Kaplan was at Brandeis University at the time) had been accused of representing a less desirable fish as a more pricey one. Kaplan analyzed extracts from the two contested fish species and showed that the muscle lactate dehydrogenase isozyme in the higher priced fish was in fact the electrophoretic form that typified the cheaper species—an early courtroom triumph for molecular biology as forensics, long before the advent of DNA fingerprinting and the noble Innocence Project. Meanwhile, at the University of Chicago, the geneticist Richard Lewontin teamed up with Jack Hubby to use starch gel electrophoresis to resolve molecular variants of certain enzymes among different fruit fly populations, instantly lifting the science of population genetics onto an entire new plane. Their pioneering work gave new meaning to Theodosius Dobzhansky’s definition of genetics (the best ever uttered in my opinion) as "the physiology of inheritance and variation" (2)
.
The early versions of protein electrophoresis could resolve proteins only to the extent that each species had a distinctive isoelectric point (with or without complexed other molecules that were often not dislodged by the methods of preparation then employed) combined with the resolving power of the particular version of the method. Unfortunately, it soon became apparent that most of the protein mixtures people wanted to resolve contained species with closely overlapping isoelectric points. I once gave a talk about "acidic" proteins in the nucleus after which a distinguished Northwestern University biochemist (and later good friend), David Shemin, asked me: "Why do you keep referring to this class of proteins as acidic? Most all proteins are acidic." Of course we investigators of chromatin proteins were using the term at that time to differentiate these proteins from the more well-known, basic histones. At the time of Shemin’s question (1974 or so) I did not know that most proteins are acidic, having been trained as a cytologist/cell biologist. But I did not feel like a fool at the podium because Shemin had asked the question in a nice, playful way, as I later came to know was his style.
Today, we all know that SDS and SDS-PAGE truly revolutionized the separation of complex protein mixtures, often boiled up from total cells or viruses (so much for the classical era of protein chemistry and purification). How this came to be is the subject of this Milestone.
Figure 1
shows the first published SDS-gel in the literature, resolving the four major capsid proteins of poliovirus (3)
. This paper was also the first to use a device for analyzing radioactive proteins after SDS-gel electrophoresis (more on this to follow). Less than a year later a second paper appeared, in which these methods were applied to a study of immunoglobulin synthesis (4)
. How did this revolution come about? The answer, ironically, sat not so much in the field of protein chemistry but rather in a particular team assembled at NIH by a visionary infectious disease pioneer, Harry Eagle. Working initially on syphilis and then turning, more broadly, to the use of cultured mammalian cells to study virus replication, Eagle’s magnetic brilliance had attracted a number of extremely talented postdocs who subsequently moved with him to the Albert Einstein College of Medicine in the early 1960s. Among them were Jacob Maizel and Matthew Scharff. The cell biology department Eagle set up at Einstein was broadly envisioned (typical of him), but it was inescapable that virology and immunology would be major foci.
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In addition to his NIH team, Eagle also attracted others to his new department at Einstein, including a rising star of virology recruited from Canberra, Bill Joklik. It was Joklik who suggested to Maizel that the detergent sodium dodecyl sulfate could perhaps dissociate poliovirus and other viruses of interest based on work Joklik had just seen (5
, 6)
. This soon led Maizel and colleagues to their breakthrough studies (3
, 4)
followed by dozens of papers by the Einstein group in the next few years on poliovirus, adenovirus, and reovirus particle proteins. A key advantage of SDS for studying the protein components of these viruses that soon became evident was that not only was the detergent able to dissociate viral particles after boiling (either purified virus, or extracts from infected cells in which virus assembly was underway) but that the only further preparative step needed prior to electrophoresis was to simply dialyze the sample to achieve the proper buffer composition and concentration. (In some cases dialysis could even be skipped if the experiment was so designed and the sample behaved.) This relative ease of application together with the gel slicing device for analyzing radioactive proteins, mentioned in passing in the first papers (3
, 4)
and soon published (7)
, rapidly catapulted SDS-PAGE further.
As initially employed, the method gave a "sharper image" of a protein mixture, and although there was a sense that the observed resolution was to some degree correlated with molecular weight, this had not yet been established. Eladio Viñuela, a visiting scientist in the laboratory of Arnold Shapiro at NYU, had the simple but elegant idea of running some proteins of known molecular weight in one of Maizel’s SDS-gels. (The term "simple but elegant" meaning that in retrospect it seems simple but the fact is that during the winter/spring of 1964–1965, when the Einstein group was doing the poliovirus study (3)
no one did the experiment.) The result (Fig. 2
) was a breathtaking inverse correlation between mobility and protein molecular weight (8)
, which stands today as one of the most enabling published experiments in the modern era of protein science.
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No matter how uniquely centered on a particular group a given scientific breakthrough may seem to be, and there can be no doubt that the Einstein investigators were the recognized "masters of the universe" in regard to SDS gel electrophoresis at the time, one must always ask whether there were other players, or at least other contenders. In the case of SDS-PAGE there was indeed another player, and he was ahead of the Einstein group by just a few months. Grant Fairbanks, a student in Cyrus Levinthal’s lab at MIT had gotten onto SDS in 1964, during his efforts to dissociate E. coli membrane proteins. He had systematically tried everything known in the field and had come down to two finalists—dichloroacetic acid and SDS. Not wishing to watch his hands and lab coat dissolve, he wisely chose SDS and had encouraging results. He also discovered that the separation principle in SDS-gel electrophoresis is not protein net charge, but is sieving in the gel matrix due to protein extended conformation. This was revealed by the fact that when the numerous intra-chain disulfide bonds of bovine serum albumin (BSA) were reduced, the resulting unfolded protein migrated more slowly in SDS-gels than non-reduced BSA, the extended conformation serving to retard migration more than the added net anionic charge (SDS) accelerated it (G. Fairbanks, personal communication). In his thesis research, Fairbanks refined the method and even discovered the inverse relationship between mobility and molecular weight (Fig. 3
). But Fairbanks was a sole graduate student, entirely on his own in this project although Levinthal, Fairbanks and a fellow student, Ronald Reeder, devised a method for autoradiography of radioactive proteins after (non-SDS) gel electrophoresis (9)
. Fairbanks’ SDS-gel electrophoresis methods were not published until 1971, 4–5 years after the Einstein group’s papers, in the context of postdoctoral work in which he applied it to another heretofore intractable membrane, that of the human erythrocyte (10)
. Just as the pioneering Einstein group had used SDS-PAGE to revolutionize virology and accelerate the study of antibody synthesis and chain variability, Fairbanks’ contributions constituted a major development in hematology (11)
. Another of Fairbanks’ contributions was the discovery that SDS, used at lower concentrations, often elicits the action of endogenous proteases. At these concentrations the unfolded protein is open to the ravages of proteases able to operate at that detergent concentration, a factor that, unrecognized, led to some later complications in red cell membrane work of others (12)
. Fairbanks’ deliberate use of high SDS concentrations killed the proteases, another of his experimental insights. The unfolding of proteins by SDS is still studied today with continuing new dimensions (13)
.
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Within months after the three seminal papers (3
, 4
, 8)
, SDS-PAGE had become a routine method. Even though this was three decades before e-mail, word spread within days and weeks on phone lines and was also the buzz at every conference during 1965–1966. By the time I arrived in Harry Eagle’s department at Einstein to begin my postdoctoral (September, 1968), there was nothing new about SDS-PAGE and hundreds of gels were being run each week, most of them ending up being sent through one of Jake Maizel’s home-made fractionators. (He allowed me to use one without any interview or check of my credentials, saying only "If you break it every student, postdoc, and faculty member of the department will want to kill you." It was more than a sufficient warning and I was very careful.)
Only two more steps remained for the transformation of SDS-PAGE into the form widely used today, viz. the introduction of the slab gel motif to facilitate simultaneous resolution of multiple samples (14
, 15)
and a modified buffer system initially introduced to improve the solubilization and resolution of bacteriophage T4 head proteins (16)
. Later developments included the marriage of Arne Tisleius’ beloved isoelectric focusing of proteins with SDS-PAGE ("2-D" gels), immunoblots ("Westerns") and finally, the ultimate user-friendly tool, commercially available precast slab SDS gels. When the latter first came upon the scene, many of us were outraged. Like all the kits now used to do many things in molecular and cell biology, whose components are secrets to our students but accurately guessed by more experienced professors, commercially sold precast SDS gels are of course "no problemo".
As mentioned at the outset, we all run proteins on SDS gels today with little thought to the pioneers (and frankly we are equally guilty about pausing to recall the innovators of most all other techniques we use). SDS had long been in the public sector (for example, as a toothpaste additive), and had been used by Julius Marmur in Paul Doty’s lab at Harvard in the 1950s to nicely get DNA out of bacteria. We can ponder whether there is ever really "anything new under the sun" but SDS as applied to gel electrophoresis was indeed something new and strutted into the theater of protein analysis as a proud grandee. Here I have endeavored to give a general account of the origins of a powerful technique in the modern era of biomedical science, with the privilege of having had some of its masters as mentors and to thus observe, with awe, this overnight sensation.
ACKNOWLEDGMENTS
Writing this essay has properly reminded the author of his great debt to caring mentors and other major influences both in electrophoresis and cell biology during his postdoctoral training at the Albert Einstein College of Medicine: Harry Eagle, Matthew Scharff, Elliott Robbins, Jacob Maizel, Donald Summers, James Darnell, Barry Bloom, Jonathan Warner, Bernard Fields, and Marshall Horwitz. Bernie and Marshall were lost to us long before their time and are thus especially remembered. I am indebted to Walter Gratzer for pointing out that paper preceded other media for protein electrophoresis. For unpublished information I also warmly thank Grant Fairbanks, my former colleague at the Worcester Foundation and a key mentor on electrophoresis. The author’s work is supported by grant MCB-0445841 from the National Science Foundation.
FOOTNOTES
The opinions expressed in editorials, essays, letters to the editor, and other articles comprising the Up Front section are those of the authors and do not necessarily reflect the opinions of FASEB or its constituent societies. The FASEB Journal welcomes all points of view and many voices. We look forward to hearing these in the form of op-ed pieces and/or letters from its readers addressed to journals@faseb.org.
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