- Email: [email protected]
SDS polyacrylamide gel electrophoresis
REVIEWS
TIBS 25 – DECEMBER 2000
and Repair Shop, where he started to build the apparatus for people who requested it. My first published description of results obtained with the slab gels was in an article on T7 in 1972 (Ref. 12). A full description, including the use of gradient gels, was published in 1973 (Ref. 13). I continued to receive requests for the detailed plans and procedures into the 1980s, and sent out hundreds of copies. These requests finally dropped off as commercial systems, mostly based on our design, became widely available. The great success in analyzing T7 proteins meant that Jake and I never did much exploring of separations of nucleic acids in the summers he was at Brookhaven. However, shortly after we started using slab gels, we began to use polyacrylamide or agarose/polyacrylamide composite gels to analyze T7 RNAs made during infection13. The discovery of restriction enzymes that cut DNA at specific nucleotide sequences14,15 provided a great stimulus for the use of electrophoretic separation of DNAs in agarose gels, which could resolve much larger DNAs than polyacrylamide gels. Much of the early work with agarose gels used tube gels and, again, slab gels had obvious advantages. By 1973, Mike McDonell, a PhD student, and Martha Simon, a research associate, had joined our group and begun using agarose gel electrophoresis to analyze T7 DNA and its restriction fragments. The vertical slabgel configuration proved unsuitable for agarose gels, and we soon developed a horizontal configuration that supported distortion-free electrophoresis at low agarose concentrations and allowed sample wells to be formed without tearing the gel16. We developed a more con-
venient apparatus shortly after, and offered plans but did not publish a description17. Again, we sent out hundreds of copies of plans and procedures and, again, the Aquebogue Machine and Repair Shop marketed copies. Other groups developed convenient designs for horizontal agarose slab gels around this time, and eventually horizontal designs became available from many commercial sources. Other important techniques that have taken advantage of slab-gel electrophoresis include two-dimensional separations of protein mixtures, introduced by O’Farrell18; blotting procedures for identifying specific nucleic acids in complex mixtures, introduced by Southern19; and DNA sequencing by the method of Sanger and Coulson20, or of Maxam and Gilbert21 (although sequencers based on capillary electrophoresis are now taking over in high-throughput DNA sequencing centers). Slab-gel electrophoresis systems for a variety of uses are now widely available, and any lab that works with proteins or nucleic acids is almost sure to have access to one or more home-built or commercial system.
Acknowledgement Work at Brookhaven National Laboratory during the period described in the article was supported by the US Atomic Energy Commission, a predecessor to our current sponsor, the US Dept of Energy.
References 1 Studier, F.W. (1965) Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373–390 2 Summers, D.F. et al. (1965) Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Natl. Acad. Sci. U. S. A. 54, 505–513 3 Maizel, J.V., Jr (1966) Acrylamide-gel electrophorograms by mechanical fractionation: radioactive adenovirus proteins. Science 151, 988–990
SDS polyacrylamide gel electrophoresis In 1961 I joined Harry Eagle’s newly created Department of Cell Biology at the Albert Einstein College of Medicine in the Bronx, New York, as an Assistant Professor. While studying poliovirus and adenovirus capsid proteins by biophysical methods, I was surprised to find four proteins1,2 after dissociating poliovirus and doing electrophoresis in 8 M ureacontaining polyacrylamide tube gels3.
590
This was unexpected because simple icosahedral RNA viruses were proposed, on theoretical grounds, to have capsids with a single protein4,5 and several small icosahedral plant viruses were known to conform to that model. Another arrival in the Department was Bill Joklik from Canberra, Australia. Bill suggested I look at the work of Laver6 who used sodium dodecyl sulfate (SDS)
4 Shapiro, A.L. et al. (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815–820 5 Studier, F.W. and Maizel, J.V., Jr (1969) T7-directed protein synthesis. Virology 39, 575–586 6 Studier, F.W. (1969) The genetics and physiology of bacteriophage T7. Virology 39, 562–574 7 Maizel, J.V., Jr (1971) Polyacrylamide gel electrophoresis of viral proteins. Methods Virol. 5, 179–246 8 Fairbanks, G., Jr et al. (1965) Analysis of 14C-labeled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 20, 393–399 9 Reid, M.S. and Bieleski, R.L. (1968) A simple apparatus for vertical flat-sheet polyacrylamide gel electrophoresis. Anal. Biochem. 22, 374–381 10 Maizel, J.V., Jr et al. (1968) The polypeptides of adenovirus 1. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36, 115–125 11 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 12 Studier, F.W. (1972) Bacteriophage T7. Science 176, 367–376 13 Studier, F.W. (1973) Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237–248 14 Kelly, T.J., Jr and Smith, H.O. (1970) A restriction enzyme from Hemophilus influenzae. II. Base sequence of the recognition site. J. Mol. Biol. 51, 393–409 15 Danna, K. and Nathans, D. (1971) Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. U. S. A. 68, 2913–2917 16 McDonell, M.W. et al. (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 110, 119–146 17 Rosenberg, A.H. et al. (1979) Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA. J. Mol. Biol. 135, 907–915 18 O’Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021 19 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517 20 Sanger, F. and Coulson, A.R. (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94, 441–448 21 Maxam, A.M. and Gilbert, W. (1977) A new method for sequencing DNA. Proc. Natl. Acad. Sci. U. S. A. 74, 560–564 22 Dunn, J.J. and Studier, F.W. (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477–535
F. WILLIAM STUDIER Biology Dept, Brookhaven National Laboratory, Upton, NY 11973, USA. Email: [email protected]
to dissociate influenza virion proteins for separation by cellulose acetate strip electrophoresis. I tried dissociating adenovirions with SDS and separating them on the high-pH, discontinuous buffer system7 using 3–5% polyacrylamide gels containing SDS. Figure 1a shows my first experiment with SDS on a 3% polyacrylamide gel stained with Amido Black. More sensitive detection of protein bands resulted from staining with Coomassie Brilliant Blue8, a dye originally used in Australia for dying wool that became widespread for gel electrophoresis. But in spite of the interesting adenovirus results, there was a nagging reproducibility problem that I later understood, but which caused me to explore other systems.
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
PII: S0968-0004(00)01693-5
REVIEWS
TIBS 25 – DECEMBER 2000 At about that time, Don Summers joined the new group headed by Jim Darnell in the Biochemistry Department at the Einstein. He was interested in studying poliovirus-infected cell proteins. We talked about SDS electrophoresis in various gels, and for various reasons Don tried electrophoresis in phosphate-buffered SDS agarose gels. As I recall, the proteins were not well resolved, and I suggested that we try some of his samples on polyacrylamide gels. These contained relatively high ionic strength (0.1 M), neutral pH, sodium phosphate buffer with 0.1% SDS in 5% polyacrylamide gel and the same high ionic strength buffer in the electrode chambers. By applying the sample, which had been heated in SDS and mercaptoethanol to unfold metastable proteins, in much lower ionic strength (0.01 M), it was possible to get quite good stacking of the sample into a narrow starting zone. This was due to two factors, the large voltage gradient across the less conductive sample and a ‘traffic jam’ effect as the SDS-treated proteins collided with the top of the gel. An example of adenovirus type 2 protein stained with Coomassie Brilliant Blue is shown in Fig. 1b. These gels were very reproducible, but another problem to be solved was detecting the viral proteins, which were mixed with all of the cellular proteins. Don and Sheldon Penman devised a method using cells infected with a high multiplicity of virus in the presence of guanidinium chloride. This suppressed viral replication but allowed the virus to shut off host protein synthesis. Viral protein synthesis could be monitored by applying short pulses of radioactive amino acids followed by chases with nonradioactive amino acids. We then needed a way to measure radioactivity in the gels. I had already been using a technique in which the cylindrical gels were teased out of the glass tubes and cut into ~100 transverse slices with a razor blade. Each slize was homogenized in a tissue homogenizer and dried onto a planchet for measuring b decay in an automated counter. Although successful, this was laborious. After my technician quit, and while thinking about the problem on a long automobile trip, I hit upon the idea of extruding the cylindrical gel through a small orifice into a flowing stream of water and onto the counting vessels. As soon as I got back to the lab I made a prototype that showed the idea would work and had the machine shop make a working model of the gel
fractionator as well as an especially designed fraction collector9,10. At that time we were collaborating with a number of people at Einstein on protein studies. Arnie Shapiro from New York University (NYU) was working on immunoglobulin synthesis in Matt Sharff’s lab just down the hall and was aware that I had done SDS-sucrose gradient centrifugation of adenovirion proteins followed by SDS gel electrophoresis of the fractions. There was a clear effect of size on mobility. Faster-sedimenting larger proteins migrated slower in electrophoresis than slowersedimenting, smaller proteins. Eladio Vinuela, in Severo Ochoa’s laboratory at NYU with whom Arnie had discussed SDS gels, then did the marvelously simple and insightful experiment of measuring the SDS gel mobility of a number of pure proteins of known molecular weight. The result was the discovery that mobility was inversely related to the logarithm of the molecular weight of the polypeptide12. This observation greatly extended the usefulness of SDS gel electrophoresis. We could now determine the number of types, sizes and the amounts of proteins in almost any biological sample. With these tools in hand we were able to make the remarkable discovery that poliovirus proteins are made as 12 large precursor molecules , and that proteolytic processing is involved in producing the mature viral proteins in the virion and the cell. Figure 2 shows the comparison of 14C virion proteins and 3H-infected cell proteins in the same gel. This was the first time that all of the proteins encoded by a genome had been seen, and that post-translational, proteolytic cleavage was recognized as an important phenomenon in virology. Don and I continued to collaborate on poliovirus and, separately, used similar strategies and variations to study other systems. A number of other colleagues at the Einstein and elsewhere made great use of these gel technologies and other peripheral developments for a wide variety of important scientific problems. Out of a desire to understand molecules as they are organized into native complexes rather than dissociated into polypeptide chains I went on sabbatical leave in 1969 to Aaron Klug’s MRC laboratory in Cambridge, UK, to learn some electron microscopy. Aaron and John Finch did outstanding work on virion structure by electron microscopy. Jonathan King was already in the lab,
and Uli Laemmli, who had recently completed his doctorate in Kellenberger’s lab in Geneva, Switzerland, arrived at the same time as I. Uli, who had done electrophoresis using 8 M urea in the high-pH disc gel system for his doctoral research, asked me about using SDS in that system, and I told of my experience with reproducibility using SDS in the disc gel system. In spite of my only partially successful experience with SDS in the disc buffer system, Uli persisted and had great success with dissociating T4 phage proteins with SDS and running them on SDS-containing gels with the discontinuous buffer system7. Uli used 10% polyacrylamide for the studies on maturation cleavage of T4 phage-head
Figure 1 SDS polyacrylamide gel electrophoresis of adenovirus type 2. (a) The first gel (7 July 1963) in which SDS was used. Adenovirus type 2 was dissociated with SDS and run on a 3% polyacrylamide gel containing 0.25% SDS in the Davis and Ornstein, high-pH, discontinuous buffer system, and stained with Amido Black. (b) Adenovirus type II dissociated with SDS and b-mercaptoethanol, run on a 0.1 M phosphatebuffered, 5% polyacrylamide gel containing 1% SDS, and stained with Coomassie Brilliant Blue. This gel is typical of the results from March 1965 and onwards. The anode is at the bottom of both gels.
591
REVIEWS
TIBS 25 – DECEMBER 2000 Maizelref.html. The ability of SDS gel electrophoresis to dissociate almost any protein complex and to resolve individual protein chains by size has proven applicable to a wide range of biochemical problems, and the method is indispensable for any lab studying proteins.
References
Ti BS
Figure 2 The simultaneous resolution and comparison by double isotopic labeling of poliovirus capsid proteins (14C) and noncapsid cellular proteins (3H) using the 0.1 M phosphate-buffered gel. For the first time, this identified all the proteins encoded by a genome, and other gels showed for the first time that proteolytic cleavage was an integral part of virus replication. The anode is on the right. Reproduced, with permission, from Ref. 12.
proteins, described in the widely quoted paper (cited more than 155 000 times as of August 2000)13. In that paper Uli referred to a joint paper in preparation on further details of the method, in which we explored the mobility of various proteins on various gel concentrations and sample concentrations (however, this manuscript was never submitted). We found that proteins of 60 kD or less migrated together at the discontinuous boundary in SDS gels of 5% polyacrylamide, which explained the variable results I experienced earlier. As higher-percentage gels began to be used it was difficult to slide them from the glass tubes after a run for radioactive analysis, so I regarded the inexpensive tubes as disposable and simply crushed the tube between spacers with a hammer and lifted the intact gel from the glass shards. There was sometimes a little consternation when people heard about this method, but it was really quite successful. Many gel technology essentials are described in a chapter11, which also contains much of the other lore of gel electrophoresis as I practiced it in the 1960s, and references to some of the work of others. Around this time, Bill Studier introduced slab gels with autoradiography on a wide scale (Ref. 14; see p. 588 in this issue of TiBS), so we began to use them because of their convenience in
592
place of gels in glass tubes. They also allowed us to use disc buffered SDS gels with gradients of polyacrylamide ranging from 7.5 to 30%. These provided optimal separation of both high- and lowmolecular-weight proteins, and enabled us to resolve early adenovirus proteins of infected cells even though cellular protein synthesis continued15. For some time we continued to use tubular gels and fractionation to compare two different samples in the same gel using double isotopic labeling, or to recover proteins from the gels. The gel electrophoresis experience would not have been possible without a mentor like Harry Eagle on whose grants I was supported during the whole early phase of gel technology development. I fondly recall his unlikely tolerance for the contraptions I was making at the time and the complete independence he allowed me. The rapid development of SDS gel technology was due to the outstanding scientists at the Einstein and elsewhere who showed me challenging problems and used the methods in important research, thereby proving their utility. This short, personal recollection omits specific reference to most of the papers and discoveries generated by fellows in my group and many close collaborators. Those references can be seen in my bibliography at http://www.lecb.ncifcrf.gov/~jmaizel/
1 Maizel, J.V., Jr (1963) Evidence for multiple components in the structural protein of Type 1 poliovirus. Biochem. Biophys. Res. Commun. 13, 483–489 2 Maizel, J.V., Jr (1964) Preparative electrophoresis of proteins in acrylamide gels. New York Academy Symposium on Gel Electrophoresis 121, 382–390 3 Reisfeld, R.A. et al. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature 195, 281 4 Crick, F.H.C. and Watson, J.D. (1957) Virus structure: general principles. CIBA Foundation Symposium on The Nature of Viruses 5, 5–13 5 Crick, F.H.C. and Watson, J.D. (1956) Structure of small viruses. Nature 177, 473–475 6 Laver, W.G. (1964) Structural studies on the protein subunits from three strains of influenza virus. J. Mol. Biol. 9, 109–124 7 Davis, B.J. (1964) Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404–427 8 Fazekas de St. Groth, S. et al. (1963) Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochem. Biophys. Acta 71, 377–391 9 Maizel, J.V., Jr (1966) Acrylamide gel electropherograms by mechanical fractionation. Radioactive adenovirus proteins. Science 151, 988–990 10 Maizel, J.V., Jr (1971) Polyacrylamide gel electrophoresis of viral proteins. Methods Virol. 5, 179–246 11 Shapiro, A.L. et al. (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDSpolyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815–820 12 Summers, D.F. et al. (1965) Evidence for virus-specific non-capsid proteins in poliovirus-infected HeLa cells. Proc. Natl. Acad. Sci. U. S. A. 54, 505–513 13 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680–685 14 Studier, F.W. (2000) Slab-gel electrophoresis. Trends Biochem. Sci. 25, 588–590 15 Walter, G. and Maizel, J.V., Jr (1974) The polypeptides of adenovirus IV. Detection of early and late virusinduced polypeptides and their distribution in subcellular fractions. Virology, 57, 402–408
JACOB V. MAIZEL, JR Laboratory of Experimental and Computational Biology, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD 21702, USA. Email: [email protected]
Students Did you know that you are entitled to a 50% discount on a subscription to TiBS? See the bound-in subscription order card for details
TIBS 25 – DECEMBER 2000
and Repair Shop, where he started to build the apparatus for people who requested it. My first published description of results obtained with the slab gels was in an article on T7 in 1972 (Ref. 12). A full description, including the use of gradient gels, was published in 1973 (Ref. 13). I continued to receive requests for the detailed plans and procedures into the 1980s, and sent out hundreds of copies. These requests finally dropped off as commercial systems, mostly based on our design, became widely available. The great success in analyzing T7 proteins meant that Jake and I never did much exploring of separations of nucleic acids in the summers he was at Brookhaven. However, shortly after we started using slab gels, we began to use polyacrylamide or agarose/polyacrylamide composite gels to analyze T7 RNAs made during infection13. The discovery of restriction enzymes that cut DNA at specific nucleotide sequences14,15 provided a great stimulus for the use of electrophoretic separation of DNAs in agarose gels, which could resolve much larger DNAs than polyacrylamide gels. Much of the early work with agarose gels used tube gels and, again, slab gels had obvious advantages. By 1973, Mike McDonell, a PhD student, and Martha Simon, a research associate, had joined our group and begun using agarose gel electrophoresis to analyze T7 DNA and its restriction fragments. The vertical slabgel configuration proved unsuitable for agarose gels, and we soon developed a horizontal configuration that supported distortion-free electrophoresis at low agarose concentrations and allowed sample wells to be formed without tearing the gel16. We developed a more con-
venient apparatus shortly after, and offered plans but did not publish a description17. Again, we sent out hundreds of copies of plans and procedures and, again, the Aquebogue Machine and Repair Shop marketed copies. Other groups developed convenient designs for horizontal agarose slab gels around this time, and eventually horizontal designs became available from many commercial sources. Other important techniques that have taken advantage of slab-gel electrophoresis include two-dimensional separations of protein mixtures, introduced by O’Farrell18; blotting procedures for identifying specific nucleic acids in complex mixtures, introduced by Southern19; and DNA sequencing by the method of Sanger and Coulson20, or of Maxam and Gilbert21 (although sequencers based on capillary electrophoresis are now taking over in high-throughput DNA sequencing centers). Slab-gel electrophoresis systems for a variety of uses are now widely available, and any lab that works with proteins or nucleic acids is almost sure to have access to one or more home-built or commercial system.
Acknowledgement Work at Brookhaven National Laboratory during the period described in the article was supported by the US Atomic Energy Commission, a predecessor to our current sponsor, the US Dept of Energy.
References 1 Studier, F.W. (1965) Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373–390 2 Summers, D.F. et al. (1965) Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Natl. Acad. Sci. U. S. A. 54, 505–513 3 Maizel, J.V., Jr (1966) Acrylamide-gel electrophorograms by mechanical fractionation: radioactive adenovirus proteins. Science 151, 988–990
SDS polyacrylamide gel electrophoresis In 1961 I joined Harry Eagle’s newly created Department of Cell Biology at the Albert Einstein College of Medicine in the Bronx, New York, as an Assistant Professor. While studying poliovirus and adenovirus capsid proteins by biophysical methods, I was surprised to find four proteins1,2 after dissociating poliovirus and doing electrophoresis in 8 M ureacontaining polyacrylamide tube gels3.
590
This was unexpected because simple icosahedral RNA viruses were proposed, on theoretical grounds, to have capsids with a single protein4,5 and several small icosahedral plant viruses were known to conform to that model. Another arrival in the Department was Bill Joklik from Canberra, Australia. Bill suggested I look at the work of Laver6 who used sodium dodecyl sulfate (SDS)
4 Shapiro, A.L. et al. (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815–820 5 Studier, F.W. and Maizel, J.V., Jr (1969) T7-directed protein synthesis. Virology 39, 575–586 6 Studier, F.W. (1969) The genetics and physiology of bacteriophage T7. Virology 39, 562–574 7 Maizel, J.V., Jr (1971) Polyacrylamide gel electrophoresis of viral proteins. Methods Virol. 5, 179–246 8 Fairbanks, G., Jr et al. (1965) Analysis of 14C-labeled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 20, 393–399 9 Reid, M.S. and Bieleski, R.L. (1968) A simple apparatus for vertical flat-sheet polyacrylamide gel electrophoresis. Anal. Biochem. 22, 374–381 10 Maizel, J.V., Jr et al. (1968) The polypeptides of adenovirus 1. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36, 115–125 11 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 12 Studier, F.W. (1972) Bacteriophage T7. Science 176, 367–376 13 Studier, F.W. (1973) Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237–248 14 Kelly, T.J., Jr and Smith, H.O. (1970) A restriction enzyme from Hemophilus influenzae. II. Base sequence of the recognition site. J. Mol. Biol. 51, 393–409 15 Danna, K. and Nathans, D. (1971) Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. U. S. A. 68, 2913–2917 16 McDonell, M.W. et al. (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 110, 119–146 17 Rosenberg, A.H. et al. (1979) Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA. J. Mol. Biol. 135, 907–915 18 O’Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021 19 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517 20 Sanger, F. and Coulson, A.R. (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94, 441–448 21 Maxam, A.M. and Gilbert, W. (1977) A new method for sequencing DNA. Proc. Natl. Acad. Sci. U. S. A. 74, 560–564 22 Dunn, J.J. and Studier, F.W. (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477–535
F. WILLIAM STUDIER Biology Dept, Brookhaven National Laboratory, Upton, NY 11973, USA. Email: [email protected]
to dissociate influenza virion proteins for separation by cellulose acetate strip electrophoresis. I tried dissociating adenovirions with SDS and separating them on the high-pH, discontinuous buffer system7 using 3–5% polyacrylamide gels containing SDS. Figure 1a shows my first experiment with SDS on a 3% polyacrylamide gel stained with Amido Black. More sensitive detection of protein bands resulted from staining with Coomassie Brilliant Blue8, a dye originally used in Australia for dying wool that became widespread for gel electrophoresis. But in spite of the interesting adenovirus results, there was a nagging reproducibility problem that I later understood, but which caused me to explore other systems.
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
PII: S0968-0004(00)01693-5
REVIEWS
TIBS 25 – DECEMBER 2000 At about that time, Don Summers joined the new group headed by Jim Darnell in the Biochemistry Department at the Einstein. He was interested in studying poliovirus-infected cell proteins. We talked about SDS electrophoresis in various gels, and for various reasons Don tried electrophoresis in phosphate-buffered SDS agarose gels. As I recall, the proteins were not well resolved, and I suggested that we try some of his samples on polyacrylamide gels. These contained relatively high ionic strength (0.1 M), neutral pH, sodium phosphate buffer with 0.1% SDS in 5% polyacrylamide gel and the same high ionic strength buffer in the electrode chambers. By applying the sample, which had been heated in SDS and mercaptoethanol to unfold metastable proteins, in much lower ionic strength (0.01 M), it was possible to get quite good stacking of the sample into a narrow starting zone. This was due to two factors, the large voltage gradient across the less conductive sample and a ‘traffic jam’ effect as the SDS-treated proteins collided with the top of the gel. An example of adenovirus type 2 protein stained with Coomassie Brilliant Blue is shown in Fig. 1b. These gels were very reproducible, but another problem to be solved was detecting the viral proteins, which were mixed with all of the cellular proteins. Don and Sheldon Penman devised a method using cells infected with a high multiplicity of virus in the presence of guanidinium chloride. This suppressed viral replication but allowed the virus to shut off host protein synthesis. Viral protein synthesis could be monitored by applying short pulses of radioactive amino acids followed by chases with nonradioactive amino acids. We then needed a way to measure radioactivity in the gels. I had already been using a technique in which the cylindrical gels were teased out of the glass tubes and cut into ~100 transverse slices with a razor blade. Each slize was homogenized in a tissue homogenizer and dried onto a planchet for measuring b decay in an automated counter. Although successful, this was laborious. After my technician quit, and while thinking about the problem on a long automobile trip, I hit upon the idea of extruding the cylindrical gel through a small orifice into a flowing stream of water and onto the counting vessels. As soon as I got back to the lab I made a prototype that showed the idea would work and had the machine shop make a working model of the gel
fractionator as well as an especially designed fraction collector9,10. At that time we were collaborating with a number of people at Einstein on protein studies. Arnie Shapiro from New York University (NYU) was working on immunoglobulin synthesis in Matt Sharff’s lab just down the hall and was aware that I had done SDS-sucrose gradient centrifugation of adenovirion proteins followed by SDS gel electrophoresis of the fractions. There was a clear effect of size on mobility. Faster-sedimenting larger proteins migrated slower in electrophoresis than slowersedimenting, smaller proteins. Eladio Vinuela, in Severo Ochoa’s laboratory at NYU with whom Arnie had discussed SDS gels, then did the marvelously simple and insightful experiment of measuring the SDS gel mobility of a number of pure proteins of known molecular weight. The result was the discovery that mobility was inversely related to the logarithm of the molecular weight of the polypeptide12. This observation greatly extended the usefulness of SDS gel electrophoresis. We could now determine the number of types, sizes and the amounts of proteins in almost any biological sample. With these tools in hand we were able to make the remarkable discovery that poliovirus proteins are made as 12 large precursor molecules , and that proteolytic processing is involved in producing the mature viral proteins in the virion and the cell. Figure 2 shows the comparison of 14C virion proteins and 3H-infected cell proteins in the same gel. This was the first time that all of the proteins encoded by a genome had been seen, and that post-translational, proteolytic cleavage was recognized as an important phenomenon in virology. Don and I continued to collaborate on poliovirus and, separately, used similar strategies and variations to study other systems. A number of other colleagues at the Einstein and elsewhere made great use of these gel technologies and other peripheral developments for a wide variety of important scientific problems. Out of a desire to understand molecules as they are organized into native complexes rather than dissociated into polypeptide chains I went on sabbatical leave in 1969 to Aaron Klug’s MRC laboratory in Cambridge, UK, to learn some electron microscopy. Aaron and John Finch did outstanding work on virion structure by electron microscopy. Jonathan King was already in the lab,
and Uli Laemmli, who had recently completed his doctorate in Kellenberger’s lab in Geneva, Switzerland, arrived at the same time as I. Uli, who had done electrophoresis using 8 M urea in the high-pH disc gel system for his doctoral research, asked me about using SDS in that system, and I told of my experience with reproducibility using SDS in the disc gel system. In spite of my only partially successful experience with SDS in the disc buffer system, Uli persisted and had great success with dissociating T4 phage proteins with SDS and running them on SDS-containing gels with the discontinuous buffer system7. Uli used 10% polyacrylamide for the studies on maturation cleavage of T4 phage-head
Figure 1 SDS polyacrylamide gel electrophoresis of adenovirus type 2. (a) The first gel (7 July 1963) in which SDS was used. Adenovirus type 2 was dissociated with SDS and run on a 3% polyacrylamide gel containing 0.25% SDS in the Davis and Ornstein, high-pH, discontinuous buffer system, and stained with Amido Black. (b) Adenovirus type II dissociated with SDS and b-mercaptoethanol, run on a 0.1 M phosphatebuffered, 5% polyacrylamide gel containing 1% SDS, and stained with Coomassie Brilliant Blue. This gel is typical of the results from March 1965 and onwards. The anode is at the bottom of both gels.
591
REVIEWS
TIBS 25 – DECEMBER 2000 Maizelref.html. The ability of SDS gel electrophoresis to dissociate almost any protein complex and to resolve individual protein chains by size has proven applicable to a wide range of biochemical problems, and the method is indispensable for any lab studying proteins.
References
Ti BS
Figure 2 The simultaneous resolution and comparison by double isotopic labeling of poliovirus capsid proteins (14C) and noncapsid cellular proteins (3H) using the 0.1 M phosphate-buffered gel. For the first time, this identified all the proteins encoded by a genome, and other gels showed for the first time that proteolytic cleavage was an integral part of virus replication. The anode is on the right. Reproduced, with permission, from Ref. 12.
proteins, described in the widely quoted paper (cited more than 155 000 times as of August 2000)13. In that paper Uli referred to a joint paper in preparation on further details of the method, in which we explored the mobility of various proteins on various gel concentrations and sample concentrations (however, this manuscript was never submitted). We found that proteins of 60 kD or less migrated together at the discontinuous boundary in SDS gels of 5% polyacrylamide, which explained the variable results I experienced earlier. As higher-percentage gels began to be used it was difficult to slide them from the glass tubes after a run for radioactive analysis, so I regarded the inexpensive tubes as disposable and simply crushed the tube between spacers with a hammer and lifted the intact gel from the glass shards. There was sometimes a little consternation when people heard about this method, but it was really quite successful. Many gel technology essentials are described in a chapter11, which also contains much of the other lore of gel electrophoresis as I practiced it in the 1960s, and references to some of the work of others. Around this time, Bill Studier introduced slab gels with autoradiography on a wide scale (Ref. 14; see p. 588 in this issue of TiBS), so we began to use them because of their convenience in
592
place of gels in glass tubes. They also allowed us to use disc buffered SDS gels with gradients of polyacrylamide ranging from 7.5 to 30%. These provided optimal separation of both high- and lowmolecular-weight proteins, and enabled us to resolve early adenovirus proteins of infected cells even though cellular protein synthesis continued15. For some time we continued to use tubular gels and fractionation to compare two different samples in the same gel using double isotopic labeling, or to recover proteins from the gels. The gel electrophoresis experience would not have been possible without a mentor like Harry Eagle on whose grants I was supported during the whole early phase of gel technology development. I fondly recall his unlikely tolerance for the contraptions I was making at the time and the complete independence he allowed me. The rapid development of SDS gel technology was due to the outstanding scientists at the Einstein and elsewhere who showed me challenging problems and used the methods in important research, thereby proving their utility. This short, personal recollection omits specific reference to most of the papers and discoveries generated by fellows in my group and many close collaborators. Those references can be seen in my bibliography at http://www.lecb.ncifcrf.gov/~jmaizel/
1 Maizel, J.V., Jr (1963) Evidence for multiple components in the structural protein of Type 1 poliovirus. Biochem. Biophys. Res. Commun. 13, 483–489 2 Maizel, J.V., Jr (1964) Preparative electrophoresis of proteins in acrylamide gels. New York Academy Symposium on Gel Electrophoresis 121, 382–390 3 Reisfeld, R.A. et al. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature 195, 281 4 Crick, F.H.C. and Watson, J.D. (1957) Virus structure: general principles. CIBA Foundation Symposium on The Nature of Viruses 5, 5–13 5 Crick, F.H.C. and Watson, J.D. (1956) Structure of small viruses. Nature 177, 473–475 6 Laver, W.G. (1964) Structural studies on the protein subunits from three strains of influenza virus. J. Mol. Biol. 9, 109–124 7 Davis, B.J. (1964) Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404–427 8 Fazekas de St. Groth, S. et al. (1963) Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochem. Biophys. Acta 71, 377–391 9 Maizel, J.V., Jr (1966) Acrylamide gel electropherograms by mechanical fractionation. Radioactive adenovirus proteins. Science 151, 988–990 10 Maizel, J.V., Jr (1971) Polyacrylamide gel electrophoresis of viral proteins. Methods Virol. 5, 179–246 11 Shapiro, A.L. et al. (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDSpolyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815–820 12 Summers, D.F. et al. (1965) Evidence for virus-specific non-capsid proteins in poliovirus-infected HeLa cells. Proc. Natl. Acad. Sci. U. S. A. 54, 505–513 13 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680–685 14 Studier, F.W. (2000) Slab-gel electrophoresis. Trends Biochem. Sci. 25, 588–590 15 Walter, G. and Maizel, J.V., Jr (1974) The polypeptides of adenovirus IV. Detection of early and late virusinduced polypeptides and their distribution in subcellular fractions. Virology, 57, 402–408
JACOB V. MAIZEL, JR Laboratory of Experimental and Computational Biology, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD 21702, USA. Email: [email protected]
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