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[17] Zone electrophoresis with polyacrylamide gel
[17]
ZONE ELECTROPHORESIS
179
a pH above their isoelectric point during dialysis, or dialyzed against dilute acetic (0.1 M) or hydrochloric (0.01 M) acid. Fractions present in a volume inconveniently large to dialyze directly may first be concentrated on a rotary evaporator. When a mechanical pump is used this can be performed at a temperature below 10 ° by cooling the condenser of the evaporator with Dry Ice and Methyl Celtosolve or acetone. The tendency of protein solutions to foam when concentrated in this manner can usually be controlled by adding a few drops of caprylic alcohol. When solutions that contain ammonium sulfate are concentrated the protein frequently precipitates as the concentration of salt increases, and can be recovered by centrifugation.
[ 17] Z o n e E l e e t r o p h o r e s i s w i t h P o l y a c r y l a m i d e G e l
By AARON M. ALTSCHUL and WILLIA• J. EVANS Introduction Polyacrylamide gel is a synthetic support medium for electrophoretic separation of proteins. 1,2 It is prepared by copolymerizing acrylamide and N,N'-methylenebisacrylamide in the apparatus in which the electrophoresis is to be carried out. This gel has properties similar to starch or other gels: superimposed on normal electrophoretic separation based on charge is the additional separation on the basis of size and shape. Polyacrylamide gel appears to offer certain advantages over other gels as a support medium in zone electrophoresis. Notable among these is the reproducibility of the patterns obtained, the apparent absence of electro-osmosis, the ability to produce transparent sheets which allows for accurate photometry, and good tensile strength which permits easy handling during staining, washing, and photometry operations. Typical electrophoretic separations are of the order of 30-minute to 3-hour duration from the time of application of the sample. The properties of the gel may be varied in two ways: by changing the proportion of monomers, and hence the amount of cross-linking, which would affect the tensile strength, and by varying the concentration to modify strength and pore size2 Polyacrylamide gel gives the same kind of separation patterns in general as obtained with other media, but apparently this gel is superior in resolving power over other gels in current use. For serum albumins, i S. R a y m o n d and L. Weintraub, Science 130, 711 (1959). 2 S. R a y m o n d and Y.-J. Wang, Anal. Biochem. 1, 391 (1960). ' M. L. White and G. H. Dorion, J. Polymer Sci. 55, 731 (1961).
180
CHAIN OR SUBUNIT SEPARATION
[17]
for example, several additional components are obtained with polyacrylamide compared to starch gel. The monomers are neurotoxins; hence reasonable precautions should be exercised. However, since the amounts of material are in general small, there is apparently no greater hazard than in the use of other common laboratory reagents. The equipment requirements for polyacrylamide gel eleetrophoresis are quite modest. Any form of a nonconducting trough, equipped for cooling, along with a D.C. power supply constitutes the requisite equipment. Several types of cell for both horizontal and vertical analytical electrophoresis are commercially available. The vertical-type cell is apparently superior in resolving power, possibly due to a chromatographic effect. Further, it is simpler to exclude air when using the vertical-type cell; this is a requisite in the polymerization process. However, where the materials migrate toward both anode and cathode, it is necessary to conduct two analytical determinations when the vertical-type cell is employed. There is an extensive literature on the use of polyacrylamide gel for analysis of protein mixturesA.5 We propose to describe, as an example, a procedure currently in use for analytical purposes based on commercially available equipment. The application of this medium for preparative electrophoresis is receiving considerable attention. In this instance, rather than providing definitive procedures we will discuss proposed methods. Some of the equipment has been commercialized but has not been in use long enough to permit assessment of performance and versatility. Equipment Figure 1 gives an example of one type of commercially available apparatus which has found rather widespread use with polyacrylamide as the support medium. There is of course no reason why other types of support media could not be used in this equipment, or why variations in design of the equipment would not be equally suitable for polyaerylamide. The sheet apparatus (Fig. 1) consists of two parts, which when put together provide space for a gel sheet 19 X 13 X 0.3 cm between two cooling surfaces. Connection with the lower electrode compartment is through a sponge; connection with the other electrode compartment is ' C. J. O. R. Morris and P. Morris, "Separation Methods in Biochemistry." Pitman, New York, 1963. a"Gel Electrophoresis," Ann. N.Y. Acad. Sci. 121, 305-650 (1964).
[17]
ZONE ELECTROPHORESIS
181
Fro. 1. Vertical gel electrophoresis equipment. Apparatus for polyacrylamide sheets: A--outer cooling plate, B--inner cooling plate, C--upper electrode, D--lower electrode, E---slots for samples, F--sponge strips supporting gel, G---levels of buffer in electrode compartments, H--buffer overflow tube [from S. Raymond, Ann. N.Y. Acad. Sci. 121, 350 (1964)]. (A commercialmodel is manufacturedby E-C Apparatus Company, Philadelphia, Pennsylvania.) by direct contact of the electrolyte with the upper end of the gel. A detailed description of the procedure with the sheet apparatus follows2 Polymerization of Gel After the apparatus is assembled, a thin gel is formed in the space between the cooling plates while the cell is in a horizontal position. A formulation that we have found convenient to use consists of Cyanogum 41 gelling agent (trade name of American Cyanamid Co. for 95% acrylamide and 5% N,N'-methylenebisacrylamide, marketed by the Fisher Scientific Co.) dissolved in phosphate buffer of pH 7.8 and ionic strength 0.03 to yield 150 ml 5% solution. This freshly made solution is filtered and to it are added the catalysts: 2.5 ml 10% solution of #-dimethylaminopropionitrile (Fisher) and 2.5 ml 10 3 solution of ammonium persulfate (Baker), freshly made, and in that order. Immediately after mixing this solution is poured into the space between the cooling plates. Care should be taken to exclude air bubbles as these inhibit or may prevent gel formation. A V-shaped slot former is inserted into the end of the gel which will be the top of the sheet when the apparatus is set 6w. J. Evans, W. B. Carney, J. M. Dechary, and A. M. Altschul, Arch. Biochem. Biophys. 96, 233 (1962).
182
CHAIN OR SUBUNIT SEPARATION
[17]
in a vertical position. Our best results were with four slots per sheet, each slot being 2.7 cm long and penetrating as a V-shaped wedge about 3 mm into the sheet. Procedure Once the gel has formed, the slot former is withdrawn and traces of monomers remaining in the slots are removed. The apparatus is then placed in a vertical position, and the electrode compartments filled with the same buffer used in formation of the gel. Samples of proteins (0.020.04 ml) are then carefully layered under the buffer solution into the slots. Although lower concentrations can be used, the preferred protein concentration is 1 3 in order to be able to observe minor components. We have found it convenient to render the protein solutions 10% with respect to dextrose to increase the density, thereby facilitating the layering procedure. A power supply capable of delivering 300 volts at 200 ma is attached to the platinum electrodes. The plates are cooled by water at 5 ° . For proteins of molecular size 20,000--300,000, 3 hours' running time at 300 volts (voltage gradient of 8 v/cm) and 180--200 ma is adequate; it would be wise to try several periods of electrophoresis in a new situation to determine the optimum duration. On completion of an experiment, the two halves of the apparatus are separated to expose the polyacrylamide sheet. This is removed from the cell and stained for approximately 5 minutes with a 1% solution of Naphthalene Black dye (Amido Black 10-B, Pfaltz and Bauer) in water-
FIG. 2. Typical electrophoresis patterns on polyacrylamide sheets: (bottom) fraction of soluble seed protein; (top) bovine serum albumin, commercial preparation. Direction of migration is from left to right.
17]
ZONE ELECTROPHORESIS
183
methanol-acetic acid (10:10:1). The excess dye is eluted from the gel by continuous washing with the same solvent mixture, after which the gel is immersed in water until it rehydrates to its original size. The gel can then be sectioned into strips and scanned in a suitable densitometer. Figure 2 shows photographs of typical patterns obtained by this technique. Disc Electrophoresis
Another analytical technique widely used is disc electrophoresisJ 's Equipment for application of this technique is commercially available (Fig. 3). In this apparatus, a multilayered polyacrylamide gel is formed: the large-pore gels are for introduction and concentration of the protein; eleetrophoretic separation takes place in the final, small-pore gel. The system for sheet electrophoresis as described is the simplest possible. Many problems in analytical electrophoresis may be handled
FIG. 3. Apparatus for disc electrophoresis [from B. J. Davis, Ann. N . Y . Acad. 121, 404 (1964)]. (A commercial model is manufactured by Canalco, Bethesda, Maryland.) Sci.
7L. Ornstein, Ann. N . Y . Acad. Sci. 121, 321 (1964). B. J. Davis, Ann. N . Y . Acad. Sci. 121, 404 (1964).
184
CHAIN OR SUBUNIT SEPARATION
[17]
in this way or by minor variations with gels in the range 4-10% concentration. It is possible to use polyacrylamide gels in the acid as well as the alkaline pH range. In addition, reagents such as urea may be used in conjunction with polyacrylamide in electrophoresis studies? The so-called discontinuous buffers can also be employed with polyacrylamide; 6,1° the effect of other complex buffers on separation and mobility has been investigated. 11 In this connection it might be well to point out that the increased resolution obtained with discontinuous and other complex buffer systems should be viewed with some caution. It is entirely possible that the increase in number of components observed under these conditions could be artifacts resulting from the binding of the protein to some of the constituents of the buffer system. In certain instances the same could be true of even simple buffer systems; acetateacetic acid buffer, for example22 Polymerization of the polyacrylamide is catalyzed by light, and this catalysis is facilitated by inclusion of riboflavin in some gel formulations. In the example given previously, polymerization takes place without added riboflavin. Fluorescent light from a lamp placed next to the apparatus is sufficient. Stains other than Naphthalene Black 12B (Amido Schwartz) may be employed. Gluten proteins were reported to stain more clearly with Nigrosin2 The gel is stained with water-soluble Nigrosin (0.0125%) in methanol-water-acetic acid (4:5:1) for 3 hours, and is destained with the same solvent followed by 5% aqueous acetic acid to restore the gel to its original size. Saphonov suggested Xylene Brilliant Cyanine G (Sandoz A. G., Basel) as being 3 times more sensitive than Amido Schwartz. This dye is dissolved (0.1%) in a mixture of methanol-water-acetic acid (10:10:1). The sheet is fixed for 20 minutes in 5% trichloroacetic acid, and washed with 4-5 portions of water. It is then stained with the dye; excess dye is removed by washing with the same solvent. 13 Akazawa et al. stained with fuchsin-sulfite for glycoproteins?* Destaining can also be accomplished by electrophoretic removal of the excess dye2 Electrolysis products will accumulate in the electrode chamber to an extent depending on the wattage and on the duration of elec9j. W. Lee, Biochim. Biophys. Acta 69, 159 (1963). loM. D. Poulik, Nature 180, 1477 (1957). 11T. G. Ferris, R. E. Easterling, and R. E. Budd, Anal. Biochem. 8, 477 (1964). 1:j, R. Cann and W. B. Goad, J. Biol. Chem. 240, 148 (1965). 18W. J. Saphonov, personal communication (1965). L4T. Akazawa, K. Saio, and N. Sugiyama, Biochem. Biophys. Res. Commun. 20, 114 (1965).
[17]
ZONE ELECTROPHORESIS
185
trophoresis. Under the conditions as described, the pH in the cathode chamber will reach values of 11 and, in the anode, 3.
Preparative Electrophoresis The high degree of discrimination achieved by eleetrophoresis of proteins on this medium has attracted interest in similar application for separation of closely related proteins, perhaps not separable by other means. There is considerable activity in this area and, while there has not yet been sufficient time to permit evaluation of any of the proposed methods under a variety of conditions, it may be valuable to review the status of the work. The simplest approach is one employed with some success on starch gel columns: locate the various bands on a sheet after electrophoresis, cut out the sections of the sheet containing the desired protein fraction, and elute by grinding in a suitable solvent. The yield has been low in those instances where such a technique was reported with polyacrylamide gels; there is always a question of contamination with fragments of the gel of the same particle size as the proteins being isolated. A second approach is to separate the protein by the usual analytical sheet electrophoresis, rotate the electric field by 90 °, and elute each of the protein fractions in a series of cups. An apparatus for accomplishing this purpose is commercially available (E-C Apparatus Co., Philadelphia, Pennsylvania). By far the greatest interest has been in a system that will allow the protein fractions successively to traverse a column, and then be washed off the bottom of the column by a stream of buffer flowing perpendicular to the electric field. The protein is prevented from entering the anode section by a suitable membrane. Several different procedures have been described, 15-18 and one device of this type (illustrated in Fig. 4) is in commercial production.19 In this particular apparatus the column through which the protein must pass is ca. 1 cm in length, and the column is doughnut-shaped to permit maximum cooling. Altschul et al. have discussed some properties of preparative electrophoresis. 2° They noted that a negatively charged material, which absorbs in the ultraviolet, comes off the gel and must be removed before ~S. Hjert~n, J. Chromatog. 11, 66 (1963). 1~j. V. Maizel, Jr., Ann. N.Y. Acad. Sci. 121, 382 (1964). ~7D. Racusen and N. Calvanico, Anal. Biochem. 7, 62 (1964). ~sU. J. Lewis and M. 0. Clark, Anal. Biochem. 6, 303 (1963). 1~T. Jovin, A. Chrambach, and M. A. Naughton, Anal. Biochem. 9, 351 (1964). 2°A. M. Altschul, W. J. Evans, W. B. Carney, E. J. McCourtney, and J. It. Brown, LiJe Sci. 3, 611 (1964).
186
CHAIN OR SUBUNIT SEPARATION
[17]
FIG. 4. Equipment for preparative polyacrylamide gel electrophoresis (Buchler Instruments, Fort Lee, New Jersey).
protein is added to prevent contamination of the protein material. Moreover, they pointed out that it is possible to reuse columns, provided the column is cooled adequately. Their first work was on a column; in later work, using an apparatus with a slit similar in principle to the one described by Maizel, 1Gthey were able to explore other properties of this system on a larger scale. One of the problems is to prevent loss by adsorption of the protein on the cellophane membrane, which becomes positively charged during electrophoresis. This can be prevented by increasing the ionic strength of the buffer flowing through the collection device. Considerable care was taken to maintain the pH constant and to remove continuously the products of electrolysis, so that the current remains constant and the ions remain the same.
ZONE ELECTROPHORESIS
179
a pH above their isoelectric point during dialysis, or dialyzed against dilute acetic (0.1 M) or hydrochloric (0.01 M) acid. Fractions present in a volume inconveniently large to dialyze directly may first be concentrated on a rotary evaporator. When a mechanical pump is used this can be performed at a temperature below 10 ° by cooling the condenser of the evaporator with Dry Ice and Methyl Celtosolve or acetone. The tendency of protein solutions to foam when concentrated in this manner can usually be controlled by adding a few drops of caprylic alcohol. When solutions that contain ammonium sulfate are concentrated the protein frequently precipitates as the concentration of salt increases, and can be recovered by centrifugation.
[ 17] Z o n e E l e e t r o p h o r e s i s w i t h P o l y a c r y l a m i d e G e l
By AARON M. ALTSCHUL and WILLIA• J. EVANS Introduction Polyacrylamide gel is a synthetic support medium for electrophoretic separation of proteins. 1,2 It is prepared by copolymerizing acrylamide and N,N'-methylenebisacrylamide in the apparatus in which the electrophoresis is to be carried out. This gel has properties similar to starch or other gels: superimposed on normal electrophoretic separation based on charge is the additional separation on the basis of size and shape. Polyacrylamide gel appears to offer certain advantages over other gels as a support medium in zone electrophoresis. Notable among these is the reproducibility of the patterns obtained, the apparent absence of electro-osmosis, the ability to produce transparent sheets which allows for accurate photometry, and good tensile strength which permits easy handling during staining, washing, and photometry operations. Typical electrophoretic separations are of the order of 30-minute to 3-hour duration from the time of application of the sample. The properties of the gel may be varied in two ways: by changing the proportion of monomers, and hence the amount of cross-linking, which would affect the tensile strength, and by varying the concentration to modify strength and pore size2 Polyacrylamide gel gives the same kind of separation patterns in general as obtained with other media, but apparently this gel is superior in resolving power over other gels in current use. For serum albumins, i S. R a y m o n d and L. Weintraub, Science 130, 711 (1959). 2 S. R a y m o n d and Y.-J. Wang, Anal. Biochem. 1, 391 (1960). ' M. L. White and G. H. Dorion, J. Polymer Sci. 55, 731 (1961).
180
CHAIN OR SUBUNIT SEPARATION
[17]
for example, several additional components are obtained with polyacrylamide compared to starch gel. The monomers are neurotoxins; hence reasonable precautions should be exercised. However, since the amounts of material are in general small, there is apparently no greater hazard than in the use of other common laboratory reagents. The equipment requirements for polyacrylamide gel eleetrophoresis are quite modest. Any form of a nonconducting trough, equipped for cooling, along with a D.C. power supply constitutes the requisite equipment. Several types of cell for both horizontal and vertical analytical electrophoresis are commercially available. The vertical-type cell is apparently superior in resolving power, possibly due to a chromatographic effect. Further, it is simpler to exclude air when using the vertical-type cell; this is a requisite in the polymerization process. However, where the materials migrate toward both anode and cathode, it is necessary to conduct two analytical determinations when the vertical-type cell is employed. There is an extensive literature on the use of polyacrylamide gel for analysis of protein mixturesA.5 We propose to describe, as an example, a procedure currently in use for analytical purposes based on commercially available equipment. The application of this medium for preparative electrophoresis is receiving considerable attention. In this instance, rather than providing definitive procedures we will discuss proposed methods. Some of the equipment has been commercialized but has not been in use long enough to permit assessment of performance and versatility. Equipment Figure 1 gives an example of one type of commercially available apparatus which has found rather widespread use with polyacrylamide as the support medium. There is of course no reason why other types of support media could not be used in this equipment, or why variations in design of the equipment would not be equally suitable for polyaerylamide. The sheet apparatus (Fig. 1) consists of two parts, which when put together provide space for a gel sheet 19 X 13 X 0.3 cm between two cooling surfaces. Connection with the lower electrode compartment is through a sponge; connection with the other electrode compartment is ' C. J. O. R. Morris and P. Morris, "Separation Methods in Biochemistry." Pitman, New York, 1963. a"Gel Electrophoresis," Ann. N.Y. Acad. Sci. 121, 305-650 (1964).
[17]
ZONE ELECTROPHORESIS
181
Fro. 1. Vertical gel electrophoresis equipment. Apparatus for polyacrylamide sheets: A--outer cooling plate, B--inner cooling plate, C--upper electrode, D--lower electrode, E---slots for samples, F--sponge strips supporting gel, G---levels of buffer in electrode compartments, H--buffer overflow tube [from S. Raymond, Ann. N.Y. Acad. Sci. 121, 350 (1964)]. (A commercialmodel is manufacturedby E-C Apparatus Company, Philadelphia, Pennsylvania.) by direct contact of the electrolyte with the upper end of the gel. A detailed description of the procedure with the sheet apparatus follows2 Polymerization of Gel After the apparatus is assembled, a thin gel is formed in the space between the cooling plates while the cell is in a horizontal position. A formulation that we have found convenient to use consists of Cyanogum 41 gelling agent (trade name of American Cyanamid Co. for 95% acrylamide and 5% N,N'-methylenebisacrylamide, marketed by the Fisher Scientific Co.) dissolved in phosphate buffer of pH 7.8 and ionic strength 0.03 to yield 150 ml 5% solution. This freshly made solution is filtered and to it are added the catalysts: 2.5 ml 10% solution of #-dimethylaminopropionitrile (Fisher) and 2.5 ml 10 3 solution of ammonium persulfate (Baker), freshly made, and in that order. Immediately after mixing this solution is poured into the space between the cooling plates. Care should be taken to exclude air bubbles as these inhibit or may prevent gel formation. A V-shaped slot former is inserted into the end of the gel which will be the top of the sheet when the apparatus is set 6w. J. Evans, W. B. Carney, J. M. Dechary, and A. M. Altschul, Arch. Biochem. Biophys. 96, 233 (1962).
182
CHAIN OR SUBUNIT SEPARATION
[17]
in a vertical position. Our best results were with four slots per sheet, each slot being 2.7 cm long and penetrating as a V-shaped wedge about 3 mm into the sheet. Procedure Once the gel has formed, the slot former is withdrawn and traces of monomers remaining in the slots are removed. The apparatus is then placed in a vertical position, and the electrode compartments filled with the same buffer used in formation of the gel. Samples of proteins (0.020.04 ml) are then carefully layered under the buffer solution into the slots. Although lower concentrations can be used, the preferred protein concentration is 1 3 in order to be able to observe minor components. We have found it convenient to render the protein solutions 10% with respect to dextrose to increase the density, thereby facilitating the layering procedure. A power supply capable of delivering 300 volts at 200 ma is attached to the platinum electrodes. The plates are cooled by water at 5 ° . For proteins of molecular size 20,000--300,000, 3 hours' running time at 300 volts (voltage gradient of 8 v/cm) and 180--200 ma is adequate; it would be wise to try several periods of electrophoresis in a new situation to determine the optimum duration. On completion of an experiment, the two halves of the apparatus are separated to expose the polyacrylamide sheet. This is removed from the cell and stained for approximately 5 minutes with a 1% solution of Naphthalene Black dye (Amido Black 10-B, Pfaltz and Bauer) in water-
FIG. 2. Typical electrophoresis patterns on polyacrylamide sheets: (bottom) fraction of soluble seed protein; (top) bovine serum albumin, commercial preparation. Direction of migration is from left to right.
17]
ZONE ELECTROPHORESIS
183
methanol-acetic acid (10:10:1). The excess dye is eluted from the gel by continuous washing with the same solvent mixture, after which the gel is immersed in water until it rehydrates to its original size. The gel can then be sectioned into strips and scanned in a suitable densitometer. Figure 2 shows photographs of typical patterns obtained by this technique. Disc Electrophoresis
Another analytical technique widely used is disc electrophoresisJ 's Equipment for application of this technique is commercially available (Fig. 3). In this apparatus, a multilayered polyacrylamide gel is formed: the large-pore gels are for introduction and concentration of the protein; eleetrophoretic separation takes place in the final, small-pore gel. The system for sheet electrophoresis as described is the simplest possible. Many problems in analytical electrophoresis may be handled
FIG. 3. Apparatus for disc electrophoresis [from B. J. Davis, Ann. N . Y . Acad. 121, 404 (1964)]. (A commercial model is manufactured by Canalco, Bethesda, Maryland.) Sci.
7L. Ornstein, Ann. N . Y . Acad. Sci. 121, 321 (1964). B. J. Davis, Ann. N . Y . Acad. Sci. 121, 404 (1964).
184
CHAIN OR SUBUNIT SEPARATION
[17]
in this way or by minor variations with gels in the range 4-10% concentration. It is possible to use polyacrylamide gels in the acid as well as the alkaline pH range. In addition, reagents such as urea may be used in conjunction with polyacrylamide in electrophoresis studies? The so-called discontinuous buffers can also be employed with polyacrylamide; 6,1° the effect of other complex buffers on separation and mobility has been investigated. 11 In this connection it might be well to point out that the increased resolution obtained with discontinuous and other complex buffer systems should be viewed with some caution. It is entirely possible that the increase in number of components observed under these conditions could be artifacts resulting from the binding of the protein to some of the constituents of the buffer system. In certain instances the same could be true of even simple buffer systems; acetateacetic acid buffer, for example22 Polymerization of the polyacrylamide is catalyzed by light, and this catalysis is facilitated by inclusion of riboflavin in some gel formulations. In the example given previously, polymerization takes place without added riboflavin. Fluorescent light from a lamp placed next to the apparatus is sufficient. Stains other than Naphthalene Black 12B (Amido Schwartz) may be employed. Gluten proteins were reported to stain more clearly with Nigrosin2 The gel is stained with water-soluble Nigrosin (0.0125%) in methanol-water-acetic acid (4:5:1) for 3 hours, and is destained with the same solvent followed by 5% aqueous acetic acid to restore the gel to its original size. Saphonov suggested Xylene Brilliant Cyanine G (Sandoz A. G., Basel) as being 3 times more sensitive than Amido Schwartz. This dye is dissolved (0.1%) in a mixture of methanol-water-acetic acid (10:10:1). The sheet is fixed for 20 minutes in 5% trichloroacetic acid, and washed with 4-5 portions of water. It is then stained with the dye; excess dye is removed by washing with the same solvent. 13 Akazawa et al. stained with fuchsin-sulfite for glycoproteins?* Destaining can also be accomplished by electrophoretic removal of the excess dye2 Electrolysis products will accumulate in the electrode chamber to an extent depending on the wattage and on the duration of elec9j. W. Lee, Biochim. Biophys. Acta 69, 159 (1963). loM. D. Poulik, Nature 180, 1477 (1957). 11T. G. Ferris, R. E. Easterling, and R. E. Budd, Anal. Biochem. 8, 477 (1964). 1:j, R. Cann and W. B. Goad, J. Biol. Chem. 240, 148 (1965). 18W. J. Saphonov, personal communication (1965). L4T. Akazawa, K. Saio, and N. Sugiyama, Biochem. Biophys. Res. Commun. 20, 114 (1965).
[17]
ZONE ELECTROPHORESIS
185
trophoresis. Under the conditions as described, the pH in the cathode chamber will reach values of 11 and, in the anode, 3.
Preparative Electrophoresis The high degree of discrimination achieved by eleetrophoresis of proteins on this medium has attracted interest in similar application for separation of closely related proteins, perhaps not separable by other means. There is considerable activity in this area and, while there has not yet been sufficient time to permit evaluation of any of the proposed methods under a variety of conditions, it may be valuable to review the status of the work. The simplest approach is one employed with some success on starch gel columns: locate the various bands on a sheet after electrophoresis, cut out the sections of the sheet containing the desired protein fraction, and elute by grinding in a suitable solvent. The yield has been low in those instances where such a technique was reported with polyacrylamide gels; there is always a question of contamination with fragments of the gel of the same particle size as the proteins being isolated. A second approach is to separate the protein by the usual analytical sheet electrophoresis, rotate the electric field by 90 °, and elute each of the protein fractions in a series of cups. An apparatus for accomplishing this purpose is commercially available (E-C Apparatus Co., Philadelphia, Pennsylvania). By far the greatest interest has been in a system that will allow the protein fractions successively to traverse a column, and then be washed off the bottom of the column by a stream of buffer flowing perpendicular to the electric field. The protein is prevented from entering the anode section by a suitable membrane. Several different procedures have been described, 15-18 and one device of this type (illustrated in Fig. 4) is in commercial production.19 In this particular apparatus the column through which the protein must pass is ca. 1 cm in length, and the column is doughnut-shaped to permit maximum cooling. Altschul et al. have discussed some properties of preparative electrophoresis. 2° They noted that a negatively charged material, which absorbs in the ultraviolet, comes off the gel and must be removed before ~S. Hjert~n, J. Chromatog. 11, 66 (1963). 1~j. V. Maizel, Jr., Ann. N.Y. Acad. Sci. 121, 382 (1964). ~7D. Racusen and N. Calvanico, Anal. Biochem. 7, 62 (1964). ~sU. J. Lewis and M. 0. Clark, Anal. Biochem. 6, 303 (1963). 1~T. Jovin, A. Chrambach, and M. A. Naughton, Anal. Biochem. 9, 351 (1964). 2°A. M. Altschul, W. J. Evans, W. B. Carney, E. J. McCourtney, and J. It. Brown, LiJe Sci. 3, 611 (1964).
186
CHAIN OR SUBUNIT SEPARATION
[17]
FIG. 4. Equipment for preparative polyacrylamide gel electrophoresis (Buchler Instruments, Fort Lee, New Jersey).
protein is added to prevent contamination of the protein material. Moreover, they pointed out that it is possible to reuse columns, provided the column is cooled adequately. Their first work was on a column; in later work, using an apparatus with a slit similar in principle to the one described by Maizel, 1Gthey were able to explore other properties of this system on a larger scale. One of the problems is to prevent loss by adsorption of the protein on the cellophane membrane, which becomes positively charged during electrophoresis. This can be prevented by increasing the ionic strength of the buffer flowing through the collection device. Considerable care was taken to maintain the pH constant and to remove continuously the products of electrolysis, so that the current remains constant and the ions remain the same.