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Single tank apparatus for slab gel and disc electrophoresis
ANALYTICAL
BIOCHEMISTRY
82,564-572
(1977)
Single Tank Apparatus for Slab Gel and Disc Electrophoresis CATHERINEDUBERTRETANDJEAN Institut de Microbiologic
LEGAULT-DEMARE
Bhtiment 409, Universitk Paris-Sud, 91405 Orsay-Cedex France
Received January 28, 1977; accepted June 14, 1977 The principle of single tank vertical gel slab electrophoresis is described. The anode buffer, brought to a density of 1.04 by the addition of sucrose, and the cathode buffer are placed above each other and separated by a layer of an insulating organic solvent of intermediate density. Following this principle, slab gel electrophoresis may be performed practically in a common beaker without the need for any specialized equipment. Three examples of easy-to-make designs are given.
Vertical slab gel electrophoresis is now used all over the world, and many different although closely related apparatuses have been put on the market. One common drawback of all these devices is the difficulty of making the seams tight enough to prevent leakage of buffer from the upper electrode compartment, but soft enough to allow assembly without breaking the glass plates. In most commercial models, these conditions are more or less fulfilled by using foam rubber or silicone rubber gaskets, with or without silicone grease. Usually, only one precisely sized model of plate can be used in a given apparatus. In addition, the larger the plates, the more difficult it becomes to prevent leaks. A partial solution to the leak problem was found in rediscovering the horizontal gel slab, as it was used originally for agar gel electrophoresis (2). However, this technique also presents some inconveniences, like the necessity of very good electric bridges, the tricky making of sample cuvettes, and the distortion of bands in thick gels. Last but not the least, commercially available equipment is usually very expensive. During the course of a study of the proteins of phage T5, we needed an inexpensive but reliable apparatus, potentially capable of accepting plates of any dimensions. Several models were constructed, based on the following principle: The anode and the cathode buffers are placed above each other in the same vessel, and separated by an insulating layer of an organic solvent of intermediate density. 564 Copyright All rights
Q 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0003.2697
APPARATUS
FOR ELECTROPHORESIS
565
FIG. 1. Model A, the simplest model designed for 85 x lOO-mm plates. The tank (height 14 cm, diameter 10 cm) is cut with a glass-cutting machine out of a I-liter Pyrex bottle. The plate is supported by a glass ring cut from the bottom of a 500~ml bottle. Rings of different heights may be obtained from a single bottle. Top buffer, 150 ml: bottom buffer, 250 ml; solvent, 550 ml. Model B, a more elaborate device composed of a Zliter Pyrex beaker for the outer tank and of an internal compartment cut out of a l-liter bottle. Four notches were made at one end of the inside cylinder to allow a larger passage to the electric current. Top buffer, 200 ml; bottom buffer, 900 ml; solvent, 600 ml. In this figure and those following, hatched areas represent top and bottom buffers.
Organic solvents used as cooling fluids in paper electrophoresis have been proposed by many authors after Cremer and Tiselius (1950), as, for example, in the different versions of the Michl apparatus (3,4). In gel electrophoresis, the use of petroleum ether was proposed by Wieme (1964). In the present case, however, the main purpose of the solvent layer is not to cool the gel, but to eliminate the need for a rigid barrier and gaskets between the electrode compartments. In the simplest model (Fig. lA), a cylindrical glass trough, just a little larger than the glass plates to be used, is filled with three liquid layers: (a) the bottom buffer, which in our experimental conditions forms the anode “compartment” and has a density brought to 1.04 by the addition of 108 g/liter of sucrose (or of any other density agent as long as it does
566
DUBERTRET
AND LEGAULT-DEMARE
not interfere with the experiment, for example, by dissolving into the solvent layer); (b) the solvent layer, which forms the insulating barrier between the two compartments and has a density usually adjusted to 1.02; and (c) the top buffer, which forms the cathode “compartment” and has a density very close to 1.00 for most of the common electrophoresis buffers. DESCRIPTION
OF THE APPARATUS
Electrodes The electrodes were made of platinum wire (0.4 mm in diameter). The top buffer electrode was usually made of a simple vertical spiral (Fig. 3a). In Models A (Fig. 1) and C (Fig. 2) it was necessary to provide a channel to allow gas to escape from the bottom compartment without going through the solvent layer. The corresponding devices are shown in Figs. 3b and 3c. Due to the existence of a central compartment, this precaution was not necessary in Model B, and a simple platinum wire, bent to a semicircular shape was used as bottom electrode (Fig. 1B). For large plates in Model C, it was necessary to use either one type-3c or two type-3b bottom electrodes, located at opposite ends of the trough, to prevent band distortion. Solvents A layer of the right density a specific gravity higher than one. A wide range of solvents criteria: The two solvents must be their solubility in water must
was obtained by mixing a solvent having 1.02 with a calculated amount of a lighter may be selected according to the following completely miscible with each other, but be as low as possible.
FIG. 2. Model C, for 10 x 20- and 20 x 20-cm plates. The tank is a Shandon Panglas Chromatank originally designed for thin-layer chromatography. Top buffer, 900 ml; bottom buffer, 600 ml; solvent, 3400 ml.
APPARATUS
FOR ELECTROPHORESIS
567
b
FIG. 3. (a) The standard electrode used as such in the top compartment. (b) Shielded standard electrode held in place inside a glass tube (25 mm in diameter) with two rubber stoppers so that gases may escape through the second hole in the stoppers. Used in Model A (one) or in Model C (two). (c) Shielded horizontal electrode used in Model C. The horizontal part of the shield is made of a glass tube cut along its axis after welding to the vertical chimney. (d) Circulating electrode used in Model C for long-run experiments. 1, bottom buffer in: 2, bottom buffer out.
They must have a very high dielectric constant. They must have a high boiling point (and/or a low volatility) in order to reduce differential evaporation and modification of the density of the mixture. They must be weakly or nontoxic and preferably nonflammable. They must be inexpensive and easily available in a reasonable state of purity. Table 1 provides some informations about the solvents which were routinely used in the present study, but many other recipes can be conceived to fit particular needs. In order to achieve maximum safety, it is advisable to carry out all the experiments under a hood. When recycled, the solvent mixture was decanted, washed with distilled water, and stored over anhydrous sodium sulfate. Its density was checked before reuse and adjusted by adding cyclohexane, the solvent which evaporated first in our case, in the amount required to bring the density to 1.02. During electrophoresis, the solvent layer became cloudy, without any noticeable effect on the separations.
568
DUBERTRET
AND LEGAULT-DEMARE
Apparatus Sizes
The dimensions of each model and the amounts of buffers and solvents they need are indicated in the legends of the figures. The three models presented here were designed to accept one or two 8.5 x IO-cm (A and B) and up to five 20 x 20-cm plates (Model C). Model A, the simplest of all three, was built first. Model B offers the advantage over Model A of eliminating the need for a shielded bottom electrode and of providing easy access to the free surface of both electrode compartments, which facilitates buffer circulation. Both models gave identical satisfactory results, but they must be considered only as examples of application of the general principle stated above. Whatever the size of the gel slab, it will stand in an almost upright position when introduced into a slightly larger cylindrical vessel. However, with very large slabs, the volumes of buffers and of solvents would rapidly become prohibitive if they were contained in a cylindrical tank. This is why we used the rectangular Model C for 20 x 20-cm gels. Several slabs may be run at the same time; their heights must be the same, but their widths may be varied at will. A glass holder made of two glass rods bent into a U-shape (thick black bars in Fig. 2) supported the lower edge of the glass plates at about 2 cm above the bottom of the tank, in TABLE SOME
RELEVANT
PROPERTIES
Tetrachloroethylene Density (g/cm3) Boiling point (“C) Melting point (“C) Vapor pressure at 20°C (mm Hg) Flammability limits in air Solubility in water (mg/lOO ml) Dielectric constant at 20°C Maximum allowable concentration in
air @pm) Mlb M2*
OF THE
SOLVENTS
0.78 81 6.5
14.0
77.5
16.3 2.3 (25°C)
USED
Cyclohexane
1.63 121 -22
Nonflammable
1
1.3-8.4% (v/v) Insoluble 2.02
14
50
39.3
100
IN THE PRESENT
Chloroform 1.50 61 -63 160 Nonflammable 750 4.80
WORKS n-Hexane 0.66 69 -95 121 1.2-6.9% (v/v) Insoluble 1.89
10
100
75
100
a The mixture of tetrachloroethylene and cyclohexane was employed for all electrophoreses. Chloroform-hexane was used only for electrophoretic elution (see text). b Ml and M2 are the proportions in volumes of the solvents in the two mixtures needed to attain a density of 1.02.
APPARATUS
FOR ELECTROPHORESIS
FIG. 4. Circulation of buffers in Model C for long-run experiments. 2, bottom buffer in; 3, bottom buffer out; 4, top buffer out.
order to allow sufficient room for the bottom volume of bottom buffer.
electrode
569
1, top buffer in;
and sufficient
Preparation of the Gel Slabs
Two glass plates separated by glass spacers and firmly pressed against each other with paper clips were held in a vertical position, with their lower edge resting on the bottom of a semicylindrical chromatography glass trough. The sides and the bottom slit were sealed by pouring a small amount of 10% polyacrylamide gelling solution along the edges of the plates and in the trough and letting it stand for 15 min. Stacking and separating gels were then poured as usual between the glass plates. After hardening of the gels, the clips were removed and the plates, held together by adherence of the gel layer, were detached from the chromatography trough and transfered into the convenient apparatus. The samples may be introduced into the starting slots before transferring the slabs into the apparatus if desired. Buffer Circulation
The amounts of buffers contained in the Model B were found sufficient for more than 3 hr of electrophoresis at 40 mA without pH modification when using the buffers described in the legend of Fig. 5. For longer times, less buffered electrolytes, or when using several 20 x 20-cm plates in Model C, it was necessary either to replace or to circulate both the top and the bottom buffers during the run.
DUBERTRET
AND LEGAULT-DEMARE
FIG. 5. Polyacrylamide electrophoresis of proteins in apparatus Model B. Top buffer (cathode compartment), Tris-chloride buffer, 0.05 M, pH 7.4, containing lOA M EDTA and 0.2% SDS; bottom buffer (anode compartment), Tris-chloride buffer, 0.05 M, pH 8.6, containing lO-3 M EDTA, 5. lOA2 M sodium acetate, and 108 g/liter of sucrose. Final density, 1.04. Solvent, tetrachloroethylenelcyclohexane (0.39/l .OO, v/v); density 1.02. Separating gel, a&amide 8%, bisacrylamide 0.24% in top buffer; stacking gel, acrylamide 5%, bisacrylamide 0.15% in top buffer. Samples: 1, chymotrypsinogen; 2, n-butanol extract of E. coli F cells; 3, capsid proteins of bacteriophage T5; 4, ovalbumin. All samples were dialysed against top buffer and extemporaneously heated for 45 set at 100°C in the presence of 10e2 M dithiothreitol. After cooling, the samples were mixed with 0.1 vol of glycerol containing 0.05% bromophenol blue as tracking dye. Electrophoresis was conducted for 7 hr at 50 V and 30 mA (about 5.5 V/cm) at room temperature.
Top buffer was displaced by slowly dropping fresh buffer in one corner of the tank, and removing it from the opposite corner through a vertical glass tube connected to a water pump, so that a constant level was maintained. Bottom buffer can be displaced exactly in the same way by using a large glass tube plunging to the bottom of the tank (Fig. 4). Another technique was also applied occasionally by using the electrode
APPARATUS
571
FOR ELECTROPHORESIS
shown in Fig. 3d; in this case, fresh buffer was circulated through cellophane tubing across the lower compartment. Buffer circulation would be greatly facilitated if it were possible to construct the apparatus in polymethacrylate and pierce holes through the sides of the outer tank. Unfortunately we were not able to find a combination of solvents which would not attack this material. Good results were occasionally obtained however with a mixture of dibenzyl ether and cyclohexane, but irregularities in the composition of commercial dibenzylether and the fact that it slowly decomposes in the presence of water and produces traces of benzaldehyde did not allow to rely upon this solvent without time-consuming purifications. Elution
of Separated
Proteins
The very same principle has been applied to the electrophoretic elution of proteins separated on acrylamide gels, using a modification of the device described by Sulitzeanu and Goldman (1965). The gel pieces containing proteins were crushed and placed between two glass wool plugs in an open-rimmed glass tube equipped with a small tight-fitting cellophane bag at its lower tip. The tube was suspended in any one of the described apparatuses, and the thickness of the solvent layer was adjusted so that the upper end of the tube was in the top buffer, while the cellophane bag was completely immersed into the bottom buffer. In this type of experiment, it was found advantageous to use a volatile solvent mixture so as to quickly get rid of the excess of solvent trapped on the outside of the cellophane bag when taking it out of the apparatus. A chloroform-n-hexane mixture was used throughout the elution experiments (see Table 1). The following examples, dealing only with overall recovery of fractionated proteins, show that an 80% yield can be obtained. (a) 14C-Labeled vesicular stomatitis virus proteins were denatured by heating for 2 min at 100°C in the presence of 1% sodium dodecylsulfate and 1OW M dithiothreitol. They were separated by electrophoresis in 10% acrylamide, on a 20 x 20-cm gel slab, at 10 V/cm for 6 hr. One track was used as a control and stained with Coomassie brilliant blue R 250. The gel in the other tracks was cut in fragments corresponding to the colored bands, and the fragments were pooled, crushed, and subjected to electrophoresis elution. The applied voltage was 10 V/cm for 5 hr; such a long time may not be necessary, but this point has not yet been further examined. The liquid in the glass tube was removed from the top with a Pasteur pipet, the cellophane bag was punctured, and radioactivity was counted in the eluate: Input radioactivity Volume of the eluate
24,720 cpm 1.20 ml
572
DUBERTRET
AND LEGAULT-DEMARE
19,110 cpm 77%
Recovered radioactivity Yield
(b) Commercial ovalbumin showed three bands when subjected to electrophoresis under the same conditions as above. The three bands were eluted together, and protein concentrations were determined by the Lowry procedure, using ovalbumin as a standard.
Input protein (pg) Volume of the eluate (ml) Recovered protein (pg) Yield (%)
Experiment
1 Experiment
2 Experiment
650 3.65 51.5 79
650 4.00 500 77
650 4.40 514 79
3
CONCLUSION
The apparatus described here may be constructed from parts available at virtually no cost in most laboratories and allows one to use a variety of sizes of gel slabs prepared according to conventional techniques. As far as a convenient tank may be available, there is no limit to the dimensions of the plates. It does not necessitate grease or gaskets of any kind. The solvent layer is very stable, inexpensive, and it does not interfere with electrophoretic separation. In our laboratory, it is routinely used with polyacrylamide and Agarose-acrylamide gels, in the presence or in the absence of dodecyl sulfate. Although primarily designed for slab gels, the same apparatus may be used for gel tubes, as demonstrated by the elution adaptation. In contrast to other widely used systems, the anode and cathode buffers cannot be regenerated by mixing because of the presence of sucrose in the bottom buffer. However, this inconvenience appears slight as compared to the many advantages offered by the simplicity of the device. ACKNOWLEDGMENTS This work was supported by funds from the Centre National de la Recherche Scientifique, D6lCgation G&&ale B la Recherche Scientifique et Technique, and Fondation pour la Recherche M&Iicale Francaise.
REFERENCES 1. 2. 3. 4. 5. 6.
Cremer, H. D., and Tiselius, A. (1950) Biochem. 2. 320,273-283. Gordon, A. H., Keil, B., and Sebesta, K. (1949) Nature (London) 164,498-499. Michl, H. (1951) Monatsschr. Chem. 82,489-493. Michl, H. (1958) f. Chromafogr. 1,93-121. Sulitzeanu, D., and Goldman, W. F. (1%5) Nature (London) 208, 1120- 1121. Wieme, R. J. (1964) Ann. N. Y. Acad. Sci. 121, 366-372.
BIOCHEMISTRY
82,564-572
(1977)
Single Tank Apparatus for Slab Gel and Disc Electrophoresis CATHERINEDUBERTRETANDJEAN Institut de Microbiologic
LEGAULT-DEMARE
Bhtiment 409, Universitk Paris-Sud, 91405 Orsay-Cedex France
Received January 28, 1977; accepted June 14, 1977 The principle of single tank vertical gel slab electrophoresis is described. The anode buffer, brought to a density of 1.04 by the addition of sucrose, and the cathode buffer are placed above each other and separated by a layer of an insulating organic solvent of intermediate density. Following this principle, slab gel electrophoresis may be performed practically in a common beaker without the need for any specialized equipment. Three examples of easy-to-make designs are given.
Vertical slab gel electrophoresis is now used all over the world, and many different although closely related apparatuses have been put on the market. One common drawback of all these devices is the difficulty of making the seams tight enough to prevent leakage of buffer from the upper electrode compartment, but soft enough to allow assembly without breaking the glass plates. In most commercial models, these conditions are more or less fulfilled by using foam rubber or silicone rubber gaskets, with or without silicone grease. Usually, only one precisely sized model of plate can be used in a given apparatus. In addition, the larger the plates, the more difficult it becomes to prevent leaks. A partial solution to the leak problem was found in rediscovering the horizontal gel slab, as it was used originally for agar gel electrophoresis (2). However, this technique also presents some inconveniences, like the necessity of very good electric bridges, the tricky making of sample cuvettes, and the distortion of bands in thick gels. Last but not the least, commercially available equipment is usually very expensive. During the course of a study of the proteins of phage T5, we needed an inexpensive but reliable apparatus, potentially capable of accepting plates of any dimensions. Several models were constructed, based on the following principle: The anode and the cathode buffers are placed above each other in the same vessel, and separated by an insulating layer of an organic solvent of intermediate density. 564 Copyright All rights
Q 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0003.2697
APPARATUS
FOR ELECTROPHORESIS
565
FIG. 1. Model A, the simplest model designed for 85 x lOO-mm plates. The tank (height 14 cm, diameter 10 cm) is cut with a glass-cutting machine out of a I-liter Pyrex bottle. The plate is supported by a glass ring cut from the bottom of a 500~ml bottle. Rings of different heights may be obtained from a single bottle. Top buffer, 150 ml: bottom buffer, 250 ml; solvent, 550 ml. Model B, a more elaborate device composed of a Zliter Pyrex beaker for the outer tank and of an internal compartment cut out of a l-liter bottle. Four notches were made at one end of the inside cylinder to allow a larger passage to the electric current. Top buffer, 200 ml; bottom buffer, 900 ml; solvent, 600 ml. In this figure and those following, hatched areas represent top and bottom buffers.
Organic solvents used as cooling fluids in paper electrophoresis have been proposed by many authors after Cremer and Tiselius (1950), as, for example, in the different versions of the Michl apparatus (3,4). In gel electrophoresis, the use of petroleum ether was proposed by Wieme (1964). In the present case, however, the main purpose of the solvent layer is not to cool the gel, but to eliminate the need for a rigid barrier and gaskets between the electrode compartments. In the simplest model (Fig. lA), a cylindrical glass trough, just a little larger than the glass plates to be used, is filled with three liquid layers: (a) the bottom buffer, which in our experimental conditions forms the anode “compartment” and has a density brought to 1.04 by the addition of 108 g/liter of sucrose (or of any other density agent as long as it does
566
DUBERTRET
AND LEGAULT-DEMARE
not interfere with the experiment, for example, by dissolving into the solvent layer); (b) the solvent layer, which forms the insulating barrier between the two compartments and has a density usually adjusted to 1.02; and (c) the top buffer, which forms the cathode “compartment” and has a density very close to 1.00 for most of the common electrophoresis buffers. DESCRIPTION
OF THE APPARATUS
Electrodes The electrodes were made of platinum wire (0.4 mm in diameter). The top buffer electrode was usually made of a simple vertical spiral (Fig. 3a). In Models A (Fig. 1) and C (Fig. 2) it was necessary to provide a channel to allow gas to escape from the bottom compartment without going through the solvent layer. The corresponding devices are shown in Figs. 3b and 3c. Due to the existence of a central compartment, this precaution was not necessary in Model B, and a simple platinum wire, bent to a semicircular shape was used as bottom electrode (Fig. 1B). For large plates in Model C, it was necessary to use either one type-3c or two type-3b bottom electrodes, located at opposite ends of the trough, to prevent band distortion. Solvents A layer of the right density a specific gravity higher than one. A wide range of solvents criteria: The two solvents must be their solubility in water must
was obtained by mixing a solvent having 1.02 with a calculated amount of a lighter may be selected according to the following completely miscible with each other, but be as low as possible.
FIG. 2. Model C, for 10 x 20- and 20 x 20-cm plates. The tank is a Shandon Panglas Chromatank originally designed for thin-layer chromatography. Top buffer, 900 ml; bottom buffer, 600 ml; solvent, 3400 ml.
APPARATUS
FOR ELECTROPHORESIS
567
b
FIG. 3. (a) The standard electrode used as such in the top compartment. (b) Shielded standard electrode held in place inside a glass tube (25 mm in diameter) with two rubber stoppers so that gases may escape through the second hole in the stoppers. Used in Model A (one) or in Model C (two). (c) Shielded horizontal electrode used in Model C. The horizontal part of the shield is made of a glass tube cut along its axis after welding to the vertical chimney. (d) Circulating electrode used in Model C for long-run experiments. 1, bottom buffer in: 2, bottom buffer out.
They must have a very high dielectric constant. They must have a high boiling point (and/or a low volatility) in order to reduce differential evaporation and modification of the density of the mixture. They must be weakly or nontoxic and preferably nonflammable. They must be inexpensive and easily available in a reasonable state of purity. Table 1 provides some informations about the solvents which were routinely used in the present study, but many other recipes can be conceived to fit particular needs. In order to achieve maximum safety, it is advisable to carry out all the experiments under a hood. When recycled, the solvent mixture was decanted, washed with distilled water, and stored over anhydrous sodium sulfate. Its density was checked before reuse and adjusted by adding cyclohexane, the solvent which evaporated first in our case, in the amount required to bring the density to 1.02. During electrophoresis, the solvent layer became cloudy, without any noticeable effect on the separations.
568
DUBERTRET
AND LEGAULT-DEMARE
Apparatus Sizes
The dimensions of each model and the amounts of buffers and solvents they need are indicated in the legends of the figures. The three models presented here were designed to accept one or two 8.5 x IO-cm (A and B) and up to five 20 x 20-cm plates (Model C). Model A, the simplest of all three, was built first. Model B offers the advantage over Model A of eliminating the need for a shielded bottom electrode and of providing easy access to the free surface of both electrode compartments, which facilitates buffer circulation. Both models gave identical satisfactory results, but they must be considered only as examples of application of the general principle stated above. Whatever the size of the gel slab, it will stand in an almost upright position when introduced into a slightly larger cylindrical vessel. However, with very large slabs, the volumes of buffers and of solvents would rapidly become prohibitive if they were contained in a cylindrical tank. This is why we used the rectangular Model C for 20 x 20-cm gels. Several slabs may be run at the same time; their heights must be the same, but their widths may be varied at will. A glass holder made of two glass rods bent into a U-shape (thick black bars in Fig. 2) supported the lower edge of the glass plates at about 2 cm above the bottom of the tank, in TABLE SOME
RELEVANT
PROPERTIES
Tetrachloroethylene Density (g/cm3) Boiling point (“C) Melting point (“C) Vapor pressure at 20°C (mm Hg) Flammability limits in air Solubility in water (mg/lOO ml) Dielectric constant at 20°C Maximum allowable concentration in
air @pm) Mlb M2*
OF THE
SOLVENTS
0.78 81 6.5
14.0
77.5
16.3 2.3 (25°C)
USED
Cyclohexane
1.63 121 -22
Nonflammable
1
1.3-8.4% (v/v) Insoluble 2.02
14
50
39.3
100
IN THE PRESENT
Chloroform 1.50 61 -63 160 Nonflammable 750 4.80
WORKS n-Hexane 0.66 69 -95 121 1.2-6.9% (v/v) Insoluble 1.89
10
100
75
100
a The mixture of tetrachloroethylene and cyclohexane was employed for all electrophoreses. Chloroform-hexane was used only for electrophoretic elution (see text). b Ml and M2 are the proportions in volumes of the solvents in the two mixtures needed to attain a density of 1.02.
APPARATUS
FOR ELECTROPHORESIS
FIG. 4. Circulation of buffers in Model C for long-run experiments. 2, bottom buffer in; 3, bottom buffer out; 4, top buffer out.
order to allow sufficient room for the bottom volume of bottom buffer.
electrode
569
1, top buffer in;
and sufficient
Preparation of the Gel Slabs
Two glass plates separated by glass spacers and firmly pressed against each other with paper clips were held in a vertical position, with their lower edge resting on the bottom of a semicylindrical chromatography glass trough. The sides and the bottom slit were sealed by pouring a small amount of 10% polyacrylamide gelling solution along the edges of the plates and in the trough and letting it stand for 15 min. Stacking and separating gels were then poured as usual between the glass plates. After hardening of the gels, the clips were removed and the plates, held together by adherence of the gel layer, were detached from the chromatography trough and transfered into the convenient apparatus. The samples may be introduced into the starting slots before transferring the slabs into the apparatus if desired. Buffer Circulation
The amounts of buffers contained in the Model B were found sufficient for more than 3 hr of electrophoresis at 40 mA without pH modification when using the buffers described in the legend of Fig. 5. For longer times, less buffered electrolytes, or when using several 20 x 20-cm plates in Model C, it was necessary either to replace or to circulate both the top and the bottom buffers during the run.
DUBERTRET
AND LEGAULT-DEMARE
FIG. 5. Polyacrylamide electrophoresis of proteins in apparatus Model B. Top buffer (cathode compartment), Tris-chloride buffer, 0.05 M, pH 7.4, containing lOA M EDTA and 0.2% SDS; bottom buffer (anode compartment), Tris-chloride buffer, 0.05 M, pH 8.6, containing lO-3 M EDTA, 5. lOA2 M sodium acetate, and 108 g/liter of sucrose. Final density, 1.04. Solvent, tetrachloroethylenelcyclohexane (0.39/l .OO, v/v); density 1.02. Separating gel, a&amide 8%, bisacrylamide 0.24% in top buffer; stacking gel, acrylamide 5%, bisacrylamide 0.15% in top buffer. Samples: 1, chymotrypsinogen; 2, n-butanol extract of E. coli F cells; 3, capsid proteins of bacteriophage T5; 4, ovalbumin. All samples were dialysed against top buffer and extemporaneously heated for 45 set at 100°C in the presence of 10e2 M dithiothreitol. After cooling, the samples were mixed with 0.1 vol of glycerol containing 0.05% bromophenol blue as tracking dye. Electrophoresis was conducted for 7 hr at 50 V and 30 mA (about 5.5 V/cm) at room temperature.
Top buffer was displaced by slowly dropping fresh buffer in one corner of the tank, and removing it from the opposite corner through a vertical glass tube connected to a water pump, so that a constant level was maintained. Bottom buffer can be displaced exactly in the same way by using a large glass tube plunging to the bottom of the tank (Fig. 4). Another technique was also applied occasionally by using the electrode
APPARATUS
571
FOR ELECTROPHORESIS
shown in Fig. 3d; in this case, fresh buffer was circulated through cellophane tubing across the lower compartment. Buffer circulation would be greatly facilitated if it were possible to construct the apparatus in polymethacrylate and pierce holes through the sides of the outer tank. Unfortunately we were not able to find a combination of solvents which would not attack this material. Good results were occasionally obtained however with a mixture of dibenzyl ether and cyclohexane, but irregularities in the composition of commercial dibenzylether and the fact that it slowly decomposes in the presence of water and produces traces of benzaldehyde did not allow to rely upon this solvent without time-consuming purifications. Elution
of Separated
Proteins
The very same principle has been applied to the electrophoretic elution of proteins separated on acrylamide gels, using a modification of the device described by Sulitzeanu and Goldman (1965). The gel pieces containing proteins were crushed and placed between two glass wool plugs in an open-rimmed glass tube equipped with a small tight-fitting cellophane bag at its lower tip. The tube was suspended in any one of the described apparatuses, and the thickness of the solvent layer was adjusted so that the upper end of the tube was in the top buffer, while the cellophane bag was completely immersed into the bottom buffer. In this type of experiment, it was found advantageous to use a volatile solvent mixture so as to quickly get rid of the excess of solvent trapped on the outside of the cellophane bag when taking it out of the apparatus. A chloroform-n-hexane mixture was used throughout the elution experiments (see Table 1). The following examples, dealing only with overall recovery of fractionated proteins, show that an 80% yield can be obtained. (a) 14C-Labeled vesicular stomatitis virus proteins were denatured by heating for 2 min at 100°C in the presence of 1% sodium dodecylsulfate and 1OW M dithiothreitol. They were separated by electrophoresis in 10% acrylamide, on a 20 x 20-cm gel slab, at 10 V/cm for 6 hr. One track was used as a control and stained with Coomassie brilliant blue R 250. The gel in the other tracks was cut in fragments corresponding to the colored bands, and the fragments were pooled, crushed, and subjected to electrophoresis elution. The applied voltage was 10 V/cm for 5 hr; such a long time may not be necessary, but this point has not yet been further examined. The liquid in the glass tube was removed from the top with a Pasteur pipet, the cellophane bag was punctured, and radioactivity was counted in the eluate: Input radioactivity Volume of the eluate
24,720 cpm 1.20 ml
572
DUBERTRET
AND LEGAULT-DEMARE
19,110 cpm 77%
Recovered radioactivity Yield
(b) Commercial ovalbumin showed three bands when subjected to electrophoresis under the same conditions as above. The three bands were eluted together, and protein concentrations were determined by the Lowry procedure, using ovalbumin as a standard.
Input protein (pg) Volume of the eluate (ml) Recovered protein (pg) Yield (%)
Experiment
1 Experiment
2 Experiment
650 3.65 51.5 79
650 4.00 500 77
650 4.40 514 79
3
CONCLUSION
The apparatus described here may be constructed from parts available at virtually no cost in most laboratories and allows one to use a variety of sizes of gel slabs prepared according to conventional techniques. As far as a convenient tank may be available, there is no limit to the dimensions of the plates. It does not necessitate grease or gaskets of any kind. The solvent layer is very stable, inexpensive, and it does not interfere with electrophoretic separation. In our laboratory, it is routinely used with polyacrylamide and Agarose-acrylamide gels, in the presence or in the absence of dodecyl sulfate. Although primarily designed for slab gels, the same apparatus may be used for gel tubes, as demonstrated by the elution adaptation. In contrast to other widely used systems, the anode and cathode buffers cannot be regenerated by mixing because of the presence of sucrose in the bottom buffer. However, this inconvenience appears slight as compared to the many advantages offered by the simplicity of the device. ACKNOWLEDGMENTS This work was supported by funds from the Centre National de la Recherche Scientifique, D6lCgation G&&ale B la Recherche Scientifique et Technique, and Fondation pour la Recherche M&Iicale Francaise.
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