Ulrich Laemmli’s Development of SDS Polyacrylamide Gel Electrophoresis
Department of Biology
Massachusetts Institute of Technology
Cambridge, MA 02139
One of the most widely used and important techniques in modern biology is SDS polyacrylamide gel electrophoresis. This technique was developed in 1970 by Ulrich K. Laemmli when he was a postdoctoral fellow with Aaron Klug in the British Medical Research Council’s Laboratory of Molecular Biology on Hills Road in Cambridge UK (Laemmli, 1970). Jacob V. Maizel Jr., who had made important contributions to the use of SDS and acrylamide gels analysis of poliovirus proteins and the use of SDS, was visiting on sabbatical (Maizel, 1964; 1966; Vinuela et al, 1967). I was also a postdoctoral fellow in the same lab and provided an additional pair of hands. The three of us were at the MRC to work with Aaron Klug, who was leading efforts to understand the structure of viruses through electron microscopy and image processing.
After the initial brief report in Laemmli’s historic 1970 Nature paper (Laemmli, 1970), Laemmli and Maizel planned to write an article laying out both the procedures and the theoretical basis of this invaluable technique. Unfortunately the article was never completed or published. In 2000 Maizel reviewed the history of the use of SDS in fractionating viral proteins, and Aaron Klug has recently published an excellent review of his and his coworker’s contributions to structural biology, but modestly neglects to mention that Laemmli SDS Page gels were developed under his watch. This article provides an account of the conditions and interactions that led to this invaluable technical development, and the scientific environment that made it possible.
Laemmli’s motivation in developing SDS gels was to analyze the structural proteins of the capsid of phage T4. In the 1960s the groups of R. H. Epstein and Edward Kellenberger in Geneva, and R. S. Edgar at Caltech had developed two classes of conditional lethal mutants of phage T4. Unlike, for example, the rII mutants, of T4, which generated large plaques, or eye color mutants of Drosophila, conditional lethal mutants could be isolated in any and every gene that encoded an essential function (Edgar and Lielausis, 1964; Epstein et al, 1968). At that time mutants of structural proteins were very rare, whether in yeast, Neurospora, flies, mice, or humans. There were two classes conditional lethals: Temperature sensitive mutants develop by Edgar (Edgar and Lielausis, 1964), and nonsense or amber mutants developed by Epstein (Epstein et al, 1968). In the latter case the mutation generated a stop codon within the normal amino acid coding region of a gene of interest. Propagating these phage strains required the presence of E. coli host strains with a mutant suppressor tRNA, which occasionally inserted an amino acid at a stop codon. In the wild type non-permissive host bacteria, the nonsense mutation resulted in premature termination of the polypeptide chain, and translation of a smaller amber fragment. Isolation of the mutants depended on their growth on one host, but not on the other. For ts mutations isolation was of phage strains that propagated at 25 or 30oC but not at 39-40oC.
Since infection of cells with nonsense or ts mutations in genes for structural proteins blocked expression of the structural protein of that gene, the result was often accumulation of morphogenetic intermediates in virus assembly. Laemmli, Kellenberger and their coworkers in Geneva had characterized the capsid-related structures accumulating in cells infected with mutants defective in head assembly (Laemmli et al, 1970a, b). As a graduate student at Caltech I had identified the intermediates in tail assembly and tail fiber assembly, working with Bob Edgar and later Bill Wood (King, 1968; King and Wood, 1969).
Discontinuous Polyacrylamide gel electrophoresis had been invented by Baruch Davis and Leonard J. Ornstein working at New York’s Mt. Sinai Hospital, in order to resolve the proteins in blood and related samples. They described their work in two important and classic papers published in the Annals of the New York Academy of Sciences (Ornstein, 1964; Davis, 1964). These articles are still worth reading. Ornstein’s paper explained how generating a sharp voltage gradient between the leading edge of one buffer and the trailing edge of a second buffer resulted in proteins of different charge forming narrow bands or discs, driven by the voltage gradient at the discontinuity. The tendency of the bands to spread was opposed by the need for a continuous voltage gradient. In these separations proteins remained native and their surface charge of their native states determined their behavior at the buffer interface. Once the proteins passed the discontinuity and entered the separating gel, they were fractionated according to charge/molecular weight.
Davis described systematic experiments searching for an optimal matrix and the reasons for settling on polyacrylamide: a) transparency; b) biologically unreactive; c) chemically inert; d) uncharged; e) controllable pore size; and f) mechanical strength. He also provided detailed instructions for preparing gels reproducibly and reliably. In the first of the pair of articles Ornstein described the development of the discontinuous gel systems. This depended on stacking and “compression” of protein species according to their isoelectric point and charge density between two different buffer systems. Ornstein described the underlying electrochemistry in detail, but full comprehension required relatively advanced understanding of solution electrochemistry.
Though Laemmli was able to isolate capsid structures from phage infected cells, he was unable to determine their protein composition, since they did not dissociate under native conditions. Jake Maizel had shown for polioviruses that the particles could be dissociated and solubilized through the use of the detergent sodium dodecyl sulfate (SDS), including SDS in the gels during fractionation. This work had led to their key discovery that in the presence of SDS, polypeptide chains migrated through acrylamide gels proportional to their molecular weight (Vinuela et al, 1967; Weber and Osborn, 1970). Maizel understood that this represented the unfolding of the polypeptide chain and coating with 1 SDS molecules/peptide. Unfortunately, in these early SDS gels, the SDS/polypeptide chain complexes migrated as broad bands. This was adequate for poliovirus with only four protein components. However, for T4, with dozens of proteins needed for particle assembly, the resolution was inadequate.
Laemmli had been educated in the Swiss technical system, and had a much deeper knowledge of electrochemistry than most of our peer molecular biologists, virologists, and geneticists. He recognized that it should be possible to get the stacking phenomena to work for an SDS polypeptide chain complex, and therefore theoretically obtain high resolution under denaturing conditions. He set about trying to find a buffer system in which the SDS/polypeptide chains would concentrate and stack at a buffer interface. This involved making up many buffer and gel solutions, casting gels in glass tubes, running samples, then cracking open the glass tubes, slicing, drying and staining the gel slices.
Since my work on T4 tail assembly was also stymied by inability to resolve the more than 20 proteins involved, I recognized the value of his goal, and the systematic nature of his approach, and offered to help in capacity of technical assistant. Laemmli was an intense and hard worker, and my memories are of breathing SDS aerosols which we sprayed on the gel tops to get flat menisci, and regular exposure to acrylamide through handling the gels for the slicing and drying, prior to staining. I’m not sure at what point we learned that acrylamide was a neurotoxin that could be absorbed through the skin, and that breathing aerosols of SDS was not the best treatment of one’s respiratory tract. Though we didn’t smoke in the midst of experiments, after a day’s work we would sit down for a cigarette. (Decades later I came down with cancer of the larynx, which I attributed to the holes punched in my vocal chord membranes by the SDS detergent aerosols, which then provided access of cigarette smoke and particles to the epithelial cells. This is the mechanism thought to account for the increased cancer of the larynx in smokers who are also heavy drinkers.)
Laemmli succeeded in finding a pair of buffers in which the SDS/polypeptide chains stacked at the discontinuity. Using this gel system he was able show that T4 heads were assembled from more than six different proteins, and identify them as the products of specific T4 genes (Laemmli 1970). Since the phenotype of mutant infected cells as visualized in the electron microscope had already been determined, he was able to define the pathway of T4 head morphogenesis (Laemmli et al, 1970). One striking feature was the proteolysis of a number of the structural proteins, within the organized lattice, coupled to the stages of icosahedral lattice transformation.
One of the proteins, the product of gene 22 was completely proteolyzed and was absent in the mature virion. This was subsequently shown to be the major scaffolding protein for T4 head assembly (Laemmli et al, 1974; Showe and Black, 1974). This scaffolding function – required for subunit assembly but removed prior to DNA packaging – was clarified when Sherwood Casjens, David Botstein and I showed that in the Phage P22 the scaffolding exited and recycled, without proteolysis (King and Casjens, 1974).
We immediately used his gel system to identify the T4 proteins needed for tail and tail fiber assembly (King and Laemmli, 1971; 1973). The 1971 paper had a more detailed description of the buffers and procedures in its Materials and Methods and was helpful to many investigators. The tail fibers also required a phage specified chaperone, the gene 57 product, and the long T4 fiber proteins failed to fold properly in its absence, and accumulated in the pellet as an inclusion body state. Laemmli further went on to demonstrate that the folding of the coat protein required a non-structural protein gp31, which we now know to be phage specific replacement of the groES subunit of GroEL/S chaperonin (Hunt et al, 1997).
The original gels were cast in tubes, which we cracked with a hammer, and then sliced the gel lengthwise for drying. Some years later William Studier and Pat O ’Farrell described slab gels, much more efficient for multiple sample than individual tube gels. This rapidly spread throughout the molecular biology community and has remained the method of choice. Coomassie Blue, derived from textile dyes, was already in use for staining proteins.
Subsequently two-dimensional gel was introduced, and of course methods like Western blotting and Northern blotting depending on the underlying stability of the acrylamide gel separation step.
Laemmli went on to make many significant contributions to our understanding of chromatin dynamics (e.g., Saitoh and Laemmli, 1994). Maizel continued to elucidate features of animal virus structure and assembly that he had started with the lower resolution SDS gels (Maizel et al, 1968a, b; Summers and Maizel, 1970). I continued to study the genetic control of phage assembly, which led to investigating folding, misfolding and aggregation of phage structural proteins (King et al, 1996). Though we had already identified the full set of 21 proteins needed to build the phage tail (King and Laemmli 1973), and the complete sequence of their interactions needed to assemble the tail (Kikuchi and King, 1975a, b, c), 30 more years passed before the structure and organization of these protein components were solved at high resolution by Rossman’s group. Similarly though the identity and function of most capsid proteins were sorted out by 1970, their conformation, interactions and detailed organization awaited the development of high-resolution cryo electron microscopy over the following three-and-a -half decades led by Wah Chiu and colleagues (Jiang et al, 2008). Only now are the full mechanistic details emerging controlling capsid assembly, DNA packaging, and DNA injection (Chen et al, 2011).
The bulk of the work Laemmli, Maizel, Klug and I carried out was funded by public agencies, in the U.S., Great Britain and Switzerland, and we never even discussed privatizing the procedures. Both the development of the technique and its rapid propagation throughout the world’s scientific community are testimony to the value of public support for biomedical research, and the existence of an open interactive international community. And of course we owe Aaron Klug and the MRC a debt of gratitude for providing a supportive, tolerant and understanding scientific environment.
Chen D-H, Matthew L. Baker, Corey F. Hryc, Frank DiMaio, Joanita Jakana, Weimin Wu, Matthew Dougherty, Cameron Haase-Pettingell, Michael F. Schmid, Wen Jiang, David Baker, Jonathan A. King, and Wah Chiu (2011). Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus. Proc. Nat.Acad. Sci., USA 108:1355-1360.
Davis, B.J. (1964) “Disc Electrophoresis. 2, Method and application to human serum proteins”. Ann. New York Acad. Sci. 121:404-427.
Edgar, R. S. and Lielausis, I (1964) Temperature sensitive mutants of bacteriophage T4D: Their isolation and genetic characterization. Genetics, 49: 649-662.
Epstein, R. H., A. Bolle, Ch. Steinberg, E. Kellenberger, E. Boy de la Tour, E. Chevalley, R.S. Edgar, M. Susman, G. H. Denhardt, and A. Lielausis (1963) Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol., 28, 375-394.
Hunt, J. F., van der Vies, S., Henry, L. and J. Diesenhofer (1997) Cell, 90: 361-371.
Jiang, W., M.L. Baker, J. Jakana, P.R. Weigele, J. King, and W. Chiu (2008). Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy. Nature 451: 1130-4.
King, J. and Laemmli, U.K. (1971) Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol., 62: 465-477.
King, J. and Laemmli, U.K. (1973) Bacteriophage T4 tail assembly: structural proteins and their genetic identification. J. Mol. Biol., 75: 315-337.
King, J. and Casjens, S. (1974) Catalytic head assembling protein in virus morphogenesis. Nature, 251: 112-119.
Laemmli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. Aug 15; 227(5259):680-5.
Laemmli UK, Mölbert E, Showe M, Kellenberger E (1970a) Form-determining function of the genes required for the assembly of the head of bacteriophage T4. J Mol Biol.;49 (1):99-113.
Laemmli UK, Beguin F, Gujer-Kellenberger G. (1970b) A factor preventing the major head protein of bacteriophage T4 from random aggregation. J Mol Biol. Jan 14;47 (1):69-85.
Laemmli UK, Teaff N, D’Ambrosia J. (1974) Maturation of the head of bacteriophage T4. III. DNA packaging into preformed heads. J Mol Biol. 88(4):749-65
Maizel, J. V., Jr. Preparative electrophoresis of proteins in acrylamide gels. Annals of the New York Academy, Symposium on Gel Electrophoresis 121(2): 382-390, 1964.
Maizel, J. V., Jr. Acrylamide gel electropherograms by mechanical fractionation. Radioactive adenovirus proteins. Science 151: 988-990, 1966.
Maizel, J.V.Jr .(2000) Review: SDS polyacrylamide gel electrophoresis. Trends in Biochemical Sciences 25: 590-592.
Maizel, J. V., Jr. and Summers, D. F.: Evidence for differences in size and composition of the poliovirus-specific polypeptides. Virology 36: 48-54, 1968.
Maizel, J. V., Jr., Summers, D. F., and Scharff, M. D.: SDS-acrylamide gel electrophoresis and its application to the proteins of poliovirus and adenovirus-infected human cells. J. Cell. Physiol. 76: 273-288, 1970.
Maizel, J. V., Jr., White, D. O., and Scharff, M. D.: The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of Types 2, 7A and 12. Virology 36: 115-125, 1968.
Maizel, J. V., Jr., White, D. O., and Scharff, M. D.: The polypeptides of adenovirus. II. Soluble proteins, cores, top components and the structure of the virion. Virology 36: 126-136, 1968.
Ornstein, L. “Disc Electrophoresis, 1, Background and Theory”. Ann New York Acad. Sci. 121:321-349 (1964),
Saitoh, Y. and Ulrich K. Laemmli (1994) Metaphase chromosome structure: Bands arise from a differential folding path of the highly AT-rich scaffold. Cell 76, pp. 609-622.
Shapiro, A. L., Vinuela, E., and Maizel, J. V., Jr.: Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28: 815-820, 1967.
Shapiro, A. L. and Maizel, J. V., Jr.: Molecular weight estimation of polypeptides by SDS-polyacrylamide gel electrophoresis: Further data concerning resolving power and general considerations. Anal. Biochem. 29: 505-514, 1969.
Summers, D. F. and Maizel, J. V., Jr. (1968) Evidence for large precursor proteins in poliovirus synthesis. Proc. Natl. Acad. Sci. USA 59: 966-971.