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A versatile apparatus for electrophoresis including isoelectrofocusing and its performances
ANALYTICAL
BIOCHEMISTRY
129, 37-45 (1983)
A Versatile Apparatus lsoelectrofocusing
for Electrophoresis Including and Its Performances’
KAZUO Biological
Institute,
Faculty
YOSHIDA
of Science, Nagoya
University,
Nagoya
464, Japan
Received May 28, 1982 A new versatile electrophoresis apparatus with a circulating cooling plate for analytical, preparative, and two-dimensional electrophoresis, including isoelectrofocusing, was designed and constructed. The apparatus consists of six main separable blocks and two removable bar electrodes. Each block in the apparatus usually has more than two roles and can easily be assembled with paper clips. Various combinations of the blocks and electrodes enable one to assemble not only an apparatus for horizontal (flat-bed), vertical slab, disc gel, and preparative electrophoresis, but also preparative isoelectrofocusing. KEY WORDS: versatile apparatus for electrophoresis and isoelectrofocusing; removable bar electrode; separable blocks. As one of the most basic separation techniques in biochemistry and molecular biology, electrophoresis has been widely accepted for both preparative and analytical purposes for proteins, enzymes, hormones, and nucleic acids (l-2). During the past 20 years, a number of new techniques in electrophoresis, including isoelectrofocusing, have been introduced in response to the increasing demands for high resolution of various samples: disc, gradient, vertical, horizontal (flat-bed), and two-dimensional gel electrophoresis and isoelectrofocusing with or without gels, and so on (l-3). In order to accommodate these new techniques of electrophoresis and isoelectrofocusing, various types of apparatus have been designed and constructed. However, each apparatus is usually constructed based on one electrophoretic technique. There are two or
three exceptions, commercially available devices of so-called multipurpose design, in spite of the fact they are only useful for horizontal electrophoresis. In addition, they are considerably expensive. So far, there is no single apparatus to satisfy all of the above electrophoretie techniques including electrofocusing. In this paper, a new, versatile apparatus for electrophoresis and isoelectrofocusing which accommodates almost all of the above techniques is described. The principle of design is to separate the apparatus into blocks or parts which usually have more than two roles in its assembly with paper clips and silicon rubber pads. In this way, the electrophoretic system can be used as an apparatus for vertical slab, horizontal, and two-dimensional gel electrophoresis and electrofocusing, as well as preparative electrophoresis and electrofocusing. Therefore, the apparatus is inexpensive because it has in one unit the function of several ’ A preliminary report in Japanese appeared in Ref. (6). devices.
0003-2697/83/030037-09$03.00/O 37
Copyright 8 1983 by Academic Press, Inc. All rights of reproduction in any form resewed
38
KAZUO
YOSHIDA
FIG. 1. Photograph of all parts and blocks for a versatile electrophoresis apparatus including a horizontal flat plate. a, electrode tray; b, horizontal electrophoresis tray or lower tray for vertical electrophoresis with a cooling plate; c, electrode tray; d, stand, side plate of lower tray, or dust cover in the case of horizontal electrophoresis: e, silicone rubber pads; f. bottom plate: g, removable bar electrodes; h, U-shaped electrode holders or tray buffer separators; i, closing acrylic plates or dust cover; j, horizontal plate.
THE
APPARATUS
Figure 1 shows all parts and blocks for the apparatus, including a horizontal plate. Each block of the apparatus was constructed from acrylic plates (5 mm thick) unless otherwise mentioned. The figure shows blocks, electrodes, plates, the horizontal plate, silicone rubber pads, bull-dog paper clips, screws, and knots. The latter three are needed for the system assembly. The figure does not contain combs for sample slots and gel molds which are common and widely used items. Figure 2a indicates how the system is assembled for horizontal electrophoresis. The arrows indicate the direction of assembly of the apparatus; a, b, and c in Fig. 2a correspond to a, b, and c in Fig. 1, respectively. Figure 2b is a photograph of the assembled apparatus. Figures 2c-e show the design drawing of a to c in Fig. 2a, respectively. The removable bar electrode consists of a power supply joint (available at an electrical parts shop), d, and a holed acrylic bar plate, a, to which platinum wire, b, was attached as
shown in Figs. 3a and b. Figure 3c shows the design drawing of the holed acrylic bar plate. The electrodes are usually immersed in the electrode tray buffer as shown in Fig. 2b, except in the case of electrofocusing. Figures 4a and b show the system assembly for vertical electrophoresis. In this case, the electrophoretic tray (b in Fig. 2a) is used as a lower buffer tray with the plate, d, and bottom plate, f. The bottom acrylic plate is 1-cm thick while the other acrylic plates are 0.5 cm thick. Figures 4c and d show the design drawing of d and fin Fig. 4a, respectively. The system is separable into two independent electrophoretic units (a and b) as shown in Fig. 5. One is a vertical electrophoresis apparatus without a cooling unit in which the upper and lower trays are bound to the stand plate (d in Fig. 4a) with paper clips. The other is a horizontal electrophoresis apparatus with open mouths on both sides in block b (in Fig. 2a) that were closed with plates and clips. The photo shows isoelectrofocusing on a flat-bed gel. In this separate use, another pair of bar electrodes is necessary.
dn
n a7
4
a4
CI:
a7 w7 -
FIG. 2. An apparatus assembled for horizontal electrophoresis and electrofocusing. (a) Schematic diagram. a, electrode buffer tray; al, hole for tube gel electrophoresis and electrofocusing; b, Bat-bed tray; bl, electrophoresis tray; b2, circulating cooling plate; b3, inlet; b4, outlet; c, electrode buffer tray. The arrows indicate the direction of assembly. (b) Photograph of (a). The photo also shows two electrode-holder guide grooves on each side of bl (see (c)). (c) Design drawing for block a in (a). (d) Design drawing for block b in (a). (e) Design drawing for block c in (a). In (c) to (d), the figures are expressed in mm and each upper and lower figure indicate the side and top view, respectively. The lower left figure in (d) is also the side view. The blocks, a to c in (a), correspond to those of Fig. 1, respectively.
40
KAZUO
YOSHIDA
a
i t or -
FIG. 3. Removable bar electrode. (a) Schematic diagram. a, acrylic bar, b, platinum wire (0.5-mm diameter); c, outlet hole for gas by electrolysis during ekctrophoresis; d, joint (commercially available at an electronic parts shop). (b) Photograph of (a). (c) Design drawing for a in (a). The figures are expressed in mm. The upper and lower figure indicate the side and top view, respectively.
Figure 6 shows another assembly as a vertical electrophoresis apparatus in which an upper and lower buffer tray were attached to the back side of the cooling plate (compare with Figs. 4a and b). Combining this assembly with that of Fig. 4, two vertical electrophoresis systems which sandwich the same cooling plate, can be assembled. In this case, one more upper tray and pairs of bar electrodes are necessary for the complete assembly. When more than two trays, b in Fig. la and Fig. 4a, are available, we can assemble longer vertical or horizontal electrophoresis apparatus by just joining the trays, b, end to end using knots, screws, and paper clips. The plate, d in Fig. 4a, was used as a stand, a dust cover for horizontal electrophoresis apparatus, or a side plate of the lower tray as described above. Direct attachment of the vertical plate, d, to the bottom plate, f, in Fig. 4a makes the construction of the apparatus easier and simpler because the four joint screws of f (except two joint screws), fl, and the horizontal plate of d, are unnecessary. This
modification has no effect on the above-mentioned roles except that of dust cover. PERFORMANCE
OF THE APPARATUS
Although the apparatus is versatile due to its characteristics as described above, only three typical performance examples with the apparatus are shown here, to save space. Figure 7a shows data on submerged electrophoresis of lambda DNA EcoRI fragments and DNA extracts of yeast cells harboring plasmid YRp17 (4) on an agarose gel in the assembled apparatus of Fig. 5b without cooling. Figure 7b shows immunoelectrophoresis of chicken lens extracts on an agarose gel. In this case, U-shaped electrode holders were used as tray-buffer separators. Figure 8a is a diagram of the system assembly for preparative electrophoresis and electrofocusing. The photograph of the preparative column assembly is shown in Fig. 8b. In electrofocusing, inlet tubing, 2 in Fig. 8a, is closed; in the case of preparative electropho-
ELECTROPHORESIS
AND
ISOELECTROFOCUSING
APPARATUS
240
;” &’ I
FIG. 4. An apparatus assembled for vertical slab electrophoresis. (a) Schematic diagram. a, upper electrode buffer tray; b, lower electrode buffer tray; d. side plate of lower tray; e. silicone rubber pad; el, slot for lower electrode: f, bottom plate; fl, joint screw. The blocks a to f correspond to a to f of Fig. I and Fig. 2, respectively. (b) Photograph of assembled apparatus of (a). (c) Design drawing for block d in (a). The upper and lower figure indicate the top and side view, respectively. (d) Design drawing for block fin (a). Top view. In (c) and (d). the figures are expressed in mm.
41
42
FIG. 5. The assembly of the apparatus as two independent units. The apparatus can be used as two independent electrophoretic units, a and b. a, for vertical slab and tube gel electrophoresis without cooling, with blocks of a, c, d, and e in Fig. 2a and Fig. 4a assembled together. b, for electrophoresis and electrofocusing on flat-bed gel, block b in Fig. 2 and two acrylic plates assembled together. The plates were used to close the open side mouth of block b. In this photo, the apparatus was set up for isoelectrofocusing on an agarose gel. b in the photo shows how the U-shaped holders fix the bar electrodes.
resis, it is opened to permit the entry of elution buffer. Especially in the case of electrofocusing, the stopper gel, 4 in Fig. 8a, is important because it stops the pressure and direct invasion of lower tray buffer. Therefore, a high concentration and/or a long polyacrylamide gel stopper tolerant to the pressure are preferable to agarose gel because easy shrinkage of agarose gel by the low pH of the electrode buffer causes the pH gradient in the column to be disturbed by the invasion of electrode buffer. On the other hand, in the case of preparative electrophoresis, the dialyzing membrane, 5, and the elution buffer system, 1 to 3 in Fig. 8a, are important, as described elsewhere ( l-2). A preparative electrofocusing profile of equine myoglobin and methyl red is shown in Fig. 9. The result clearly shows that a pH gradient was formed and then myoglobin and methyl red were separated according to their ~1. Some methyl red was precipitated at the bottom of the column. As an alternative method of fractionation, an injection needle was connected to a pump through tubing pricks on the silicon rubber joint, 3, between
the preparative column and stopper gel, 4 in Fig. 8a, and then sucked out with the pump after isoelectrofocusing. However, this is not applicable to preparative gel electrophoresis. The seven independent columns can be set up and separations carried out in the apparatus at the same time. In this apparatus, one hole of block a in Fig. 2a and 4a is large enough to permit a 3-cm-diameter column for preparative electrophoresis and electrofocusing (see Fig. 2~). The above column device for preparative electrofocusing and electrophoresis is applicable to other vertical electrophoresis devices with slight modifications. The apparatus described in this paper has several advantages. It accommodates almost all electrophoretic and electrofocusing techniques, not only horizontal, vertical, and disc gel electrophoresis, but preparative isoelectrofocusing as well as preparative electrophoresis. The apparatus is separable into two or three independent units. It is inexpensive because it is separable into several blocks and bar electrodes which usually have more than two roles and are independently useful according to the changes in their assembly.
FIG. 6. Another assembly combination for vertical slab and disc gel electrophoresis.
a
FIG. 7. Submerged electrophoresis and immunoelectrophoresis in the apparatus. (a) Submerged electrophoresis of DNA on 0.8% agarose gel. Electrophoresis was carried out in the assembled apparatus of Fig. Sb without cooling. Agarose size is 10 X 10 X 0.25 cm. Run at 80 V for 3.5 h in 40 mM Tris-HCl-20 mM sodium acetate-2 mM EDTA-0.5 &ml ethidium bromide (pH 7.8). 1, EcoRI digests of XDNA; 4-9, DNA extracts from yeast cells harboring plasmid YRpl7 (4). (b) Immunoelectrophoresis of chick lens extracts. Electrophoresis was carried out in 1.5% Difco noble agar on a glass slide (2.5 X 7.5 cm) which was placed in the apparatus. Run for 2 h at 150 V in barbital buffer (pH 8.6, I’/2 = 0.05). Antiserum application and other details were described previously in Ref. (5).
43
a
b
8
2.
7
6. 5: tb
8. An assembly for preparative isoelectrofocusing and electrophoresis. (a) Schematic diagram of preparative column. a, upper buffer tray; b, lower buffer tray; 1, reservoir of elution buffer; 2, connecting tubing; 3, silicone rubber tubing, 4, stopper gel: 5, dialyzing tubing membrane; 6, porous plastic plate; 7. polyethylene tubing; 8, glass preparative column; 9, silicone rubber connector of upper tray; 10, peristalic pump; 11, test tube. l-9 were handmade. The column 8 was submerged in the lower tray buffer b which was cooled by circulating chilled water. (b) Photograph of (a). The large and small arrows indicate the column and magnetic stirring bar, respectively. FIG.
10
0.5
8
0.4
0.3
4
0.2
2
0.1
0
10
+
20
30
40 Fraction
50 NO.
60
70
80
100
x P 2
I 2 0
0 -
FIG.9. Preparative isoelectrofocusing in the apparatus. Discontinuous density gradient in the column (1 X 20 cm) made by 5-402 sucrose in which carrier ampholyte (pH 3 to 10) (LKB) 1% was contained. Upper tray contained 0.25 N NaOH. Lower tray contained 0.3% phosphoric acid. After electrofocusing for 12 h at 350 V under the ice-cooled condition, each fraction (7 drops) was collected through a guiding tube, 7 in Fig. 8, by siphon action (in this case, the pump 10 was not used). One milliliter of distilled water was added, then pH and absorbance of each fraction were measured. Electrofocusing samples were equine skeletal muscle myoglobin and methyl red.
ELECTROPHORESIS
AND ISOELECTROFOCUSING
ACKNOWLEDGMENTS I am grateful to Dr. A. Katoh, Mercy Hospital, Pittsburgh, Pennsylvania, for his critical reading of the manuscript and helpful suggestions. I am also indebted to Mrs. G. Takamatu, K. Koizumi, and Ms. C. Miwa of the Machine Shop for their kind help during the preparation of the manuscript.
REFERENCES 1. Mauer, H. R. (1971) Disc electrophoresis, de Gruyter, Berlin/New York.
APPARATUS
45
2. Gordon, A. H. (1972) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, E., eds.), Vol. 1, Part 1, North-Holland, Amsterdam/New York. 3. Catsimpoolas, N., and Drysdale, J. (eds.) (1977) Biological and Biomedical Applications of Isoelectric Focusing, Plenum, New York. 4. Struhl, K., Cameron, J. R., and Davis, R. W. (1976) Pmt. Nat. Acad. Sci. USA 73, 1471-1475. 5. Yoshida, K., and Katoh, A. (1971) Exp. Eye Rex 11, 122-131. 6. Yoshida, K. (1981) Pro&in, Nucleic Acid Enzyme [Tanpakushitrt Kakusan Kowo] 26, 258-260. [in Japanese]
BIOCHEMISTRY
129, 37-45 (1983)
A Versatile Apparatus lsoelectrofocusing
for Electrophoresis Including and Its Performances’
KAZUO Biological
Institute,
Faculty
YOSHIDA
of Science, Nagoya
University,
Nagoya
464, Japan
Received May 28, 1982 A new versatile electrophoresis apparatus with a circulating cooling plate for analytical, preparative, and two-dimensional electrophoresis, including isoelectrofocusing, was designed and constructed. The apparatus consists of six main separable blocks and two removable bar electrodes. Each block in the apparatus usually has more than two roles and can easily be assembled with paper clips. Various combinations of the blocks and electrodes enable one to assemble not only an apparatus for horizontal (flat-bed), vertical slab, disc gel, and preparative electrophoresis, but also preparative isoelectrofocusing. KEY WORDS: versatile apparatus for electrophoresis and isoelectrofocusing; removable bar electrode; separable blocks. As one of the most basic separation techniques in biochemistry and molecular biology, electrophoresis has been widely accepted for both preparative and analytical purposes for proteins, enzymes, hormones, and nucleic acids (l-2). During the past 20 years, a number of new techniques in electrophoresis, including isoelectrofocusing, have been introduced in response to the increasing demands for high resolution of various samples: disc, gradient, vertical, horizontal (flat-bed), and two-dimensional gel electrophoresis and isoelectrofocusing with or without gels, and so on (l-3). In order to accommodate these new techniques of electrophoresis and isoelectrofocusing, various types of apparatus have been designed and constructed. However, each apparatus is usually constructed based on one electrophoretic technique. There are two or
three exceptions, commercially available devices of so-called multipurpose design, in spite of the fact they are only useful for horizontal electrophoresis. In addition, they are considerably expensive. So far, there is no single apparatus to satisfy all of the above electrophoretie techniques including electrofocusing. In this paper, a new, versatile apparatus for electrophoresis and isoelectrofocusing which accommodates almost all of the above techniques is described. The principle of design is to separate the apparatus into blocks or parts which usually have more than two roles in its assembly with paper clips and silicon rubber pads. In this way, the electrophoretic system can be used as an apparatus for vertical slab, horizontal, and two-dimensional gel electrophoresis and electrofocusing, as well as preparative electrophoresis and electrofocusing. Therefore, the apparatus is inexpensive because it has in one unit the function of several ’ A preliminary report in Japanese appeared in Ref. (6). devices.
0003-2697/83/030037-09$03.00/O 37
Copyright 8 1983 by Academic Press, Inc. All rights of reproduction in any form resewed
38
KAZUO
YOSHIDA
FIG. 1. Photograph of all parts and blocks for a versatile electrophoresis apparatus including a horizontal flat plate. a, electrode tray; b, horizontal electrophoresis tray or lower tray for vertical electrophoresis with a cooling plate; c, electrode tray; d, stand, side plate of lower tray, or dust cover in the case of horizontal electrophoresis: e, silicone rubber pads; f. bottom plate: g, removable bar electrodes; h, U-shaped electrode holders or tray buffer separators; i, closing acrylic plates or dust cover; j, horizontal plate.
THE
APPARATUS
Figure 1 shows all parts and blocks for the apparatus, including a horizontal plate. Each block of the apparatus was constructed from acrylic plates (5 mm thick) unless otherwise mentioned. The figure shows blocks, electrodes, plates, the horizontal plate, silicone rubber pads, bull-dog paper clips, screws, and knots. The latter three are needed for the system assembly. The figure does not contain combs for sample slots and gel molds which are common and widely used items. Figure 2a indicates how the system is assembled for horizontal electrophoresis. The arrows indicate the direction of assembly of the apparatus; a, b, and c in Fig. 2a correspond to a, b, and c in Fig. 1, respectively. Figure 2b is a photograph of the assembled apparatus. Figures 2c-e show the design drawing of a to c in Fig. 2a, respectively. The removable bar electrode consists of a power supply joint (available at an electrical parts shop), d, and a holed acrylic bar plate, a, to which platinum wire, b, was attached as
shown in Figs. 3a and b. Figure 3c shows the design drawing of the holed acrylic bar plate. The electrodes are usually immersed in the electrode tray buffer as shown in Fig. 2b, except in the case of electrofocusing. Figures 4a and b show the system assembly for vertical electrophoresis. In this case, the electrophoretic tray (b in Fig. 2a) is used as a lower buffer tray with the plate, d, and bottom plate, f. The bottom acrylic plate is 1-cm thick while the other acrylic plates are 0.5 cm thick. Figures 4c and d show the design drawing of d and fin Fig. 4a, respectively. The system is separable into two independent electrophoretic units (a and b) as shown in Fig. 5. One is a vertical electrophoresis apparatus without a cooling unit in which the upper and lower trays are bound to the stand plate (d in Fig. 4a) with paper clips. The other is a horizontal electrophoresis apparatus with open mouths on both sides in block b (in Fig. 2a) that were closed with plates and clips. The photo shows isoelectrofocusing on a flat-bed gel. In this separate use, another pair of bar electrodes is necessary.
dn
n a7
4
a4
CI:
a7 w7 -
FIG. 2. An apparatus assembled for horizontal electrophoresis and electrofocusing. (a) Schematic diagram. a, electrode buffer tray; al, hole for tube gel electrophoresis and electrofocusing; b, Bat-bed tray; bl, electrophoresis tray; b2, circulating cooling plate; b3, inlet; b4, outlet; c, electrode buffer tray. The arrows indicate the direction of assembly. (b) Photograph of (a). The photo also shows two electrode-holder guide grooves on each side of bl (see (c)). (c) Design drawing for block a in (a). (d) Design drawing for block b in (a). (e) Design drawing for block c in (a). In (c) to (d), the figures are expressed in mm and each upper and lower figure indicate the side and top view, respectively. The lower left figure in (d) is also the side view. The blocks, a to c in (a), correspond to those of Fig. 1, respectively.
40
KAZUO
YOSHIDA
a
i t or -
FIG. 3. Removable bar electrode. (a) Schematic diagram. a, acrylic bar, b, platinum wire (0.5-mm diameter); c, outlet hole for gas by electrolysis during ekctrophoresis; d, joint (commercially available at an electronic parts shop). (b) Photograph of (a). (c) Design drawing for a in (a). The figures are expressed in mm. The upper and lower figure indicate the side and top view, respectively.
Figure 6 shows another assembly as a vertical electrophoresis apparatus in which an upper and lower buffer tray were attached to the back side of the cooling plate (compare with Figs. 4a and b). Combining this assembly with that of Fig. 4, two vertical electrophoresis systems which sandwich the same cooling plate, can be assembled. In this case, one more upper tray and pairs of bar electrodes are necessary for the complete assembly. When more than two trays, b in Fig. la and Fig. 4a, are available, we can assemble longer vertical or horizontal electrophoresis apparatus by just joining the trays, b, end to end using knots, screws, and paper clips. The plate, d in Fig. 4a, was used as a stand, a dust cover for horizontal electrophoresis apparatus, or a side plate of the lower tray as described above. Direct attachment of the vertical plate, d, to the bottom plate, f, in Fig. 4a makes the construction of the apparatus easier and simpler because the four joint screws of f (except two joint screws), fl, and the horizontal plate of d, are unnecessary. This
modification has no effect on the above-mentioned roles except that of dust cover. PERFORMANCE
OF THE APPARATUS
Although the apparatus is versatile due to its characteristics as described above, only three typical performance examples with the apparatus are shown here, to save space. Figure 7a shows data on submerged electrophoresis of lambda DNA EcoRI fragments and DNA extracts of yeast cells harboring plasmid YRp17 (4) on an agarose gel in the assembled apparatus of Fig. 5b without cooling. Figure 7b shows immunoelectrophoresis of chicken lens extracts on an agarose gel. In this case, U-shaped electrode holders were used as tray-buffer separators. Figure 8a is a diagram of the system assembly for preparative electrophoresis and electrofocusing. The photograph of the preparative column assembly is shown in Fig. 8b. In electrofocusing, inlet tubing, 2 in Fig. 8a, is closed; in the case of preparative electropho-
ELECTROPHORESIS
AND
ISOELECTROFOCUSING
APPARATUS
240
;” &’ I
FIG. 4. An apparatus assembled for vertical slab electrophoresis. (a) Schematic diagram. a, upper electrode buffer tray; b, lower electrode buffer tray; d. side plate of lower tray; e. silicone rubber pad; el, slot for lower electrode: f, bottom plate; fl, joint screw. The blocks a to f correspond to a to f of Fig. I and Fig. 2, respectively. (b) Photograph of assembled apparatus of (a). (c) Design drawing for block d in (a). The upper and lower figure indicate the top and side view, respectively. (d) Design drawing for block fin (a). Top view. In (c) and (d). the figures are expressed in mm.
41
42
FIG. 5. The assembly of the apparatus as two independent units. The apparatus can be used as two independent electrophoretic units, a and b. a, for vertical slab and tube gel electrophoresis without cooling, with blocks of a, c, d, and e in Fig. 2a and Fig. 4a assembled together. b, for electrophoresis and electrofocusing on flat-bed gel, block b in Fig. 2 and two acrylic plates assembled together. The plates were used to close the open side mouth of block b. In this photo, the apparatus was set up for isoelectrofocusing on an agarose gel. b in the photo shows how the U-shaped holders fix the bar electrodes.
resis, it is opened to permit the entry of elution buffer. Especially in the case of electrofocusing, the stopper gel, 4 in Fig. 8a, is important because it stops the pressure and direct invasion of lower tray buffer. Therefore, a high concentration and/or a long polyacrylamide gel stopper tolerant to the pressure are preferable to agarose gel because easy shrinkage of agarose gel by the low pH of the electrode buffer causes the pH gradient in the column to be disturbed by the invasion of electrode buffer. On the other hand, in the case of preparative electrophoresis, the dialyzing membrane, 5, and the elution buffer system, 1 to 3 in Fig. 8a, are important, as described elsewhere ( l-2). A preparative electrofocusing profile of equine myoglobin and methyl red is shown in Fig. 9. The result clearly shows that a pH gradient was formed and then myoglobin and methyl red were separated according to their ~1. Some methyl red was precipitated at the bottom of the column. As an alternative method of fractionation, an injection needle was connected to a pump through tubing pricks on the silicon rubber joint, 3, between
the preparative column and stopper gel, 4 in Fig. 8a, and then sucked out with the pump after isoelectrofocusing. However, this is not applicable to preparative gel electrophoresis. The seven independent columns can be set up and separations carried out in the apparatus at the same time. In this apparatus, one hole of block a in Fig. 2a and 4a is large enough to permit a 3-cm-diameter column for preparative electrophoresis and electrofocusing (see Fig. 2~). The above column device for preparative electrofocusing and electrophoresis is applicable to other vertical electrophoresis devices with slight modifications. The apparatus described in this paper has several advantages. It accommodates almost all electrophoretic and electrofocusing techniques, not only horizontal, vertical, and disc gel electrophoresis, but preparative isoelectrofocusing as well as preparative electrophoresis. The apparatus is separable into two or three independent units. It is inexpensive because it is separable into several blocks and bar electrodes which usually have more than two roles and are independently useful according to the changes in their assembly.
FIG. 6. Another assembly combination for vertical slab and disc gel electrophoresis.
a
FIG. 7. Submerged electrophoresis and immunoelectrophoresis in the apparatus. (a) Submerged electrophoresis of DNA on 0.8% agarose gel. Electrophoresis was carried out in the assembled apparatus of Fig. Sb without cooling. Agarose size is 10 X 10 X 0.25 cm. Run at 80 V for 3.5 h in 40 mM Tris-HCl-20 mM sodium acetate-2 mM EDTA-0.5 &ml ethidium bromide (pH 7.8). 1, EcoRI digests of XDNA; 4-9, DNA extracts from yeast cells harboring plasmid YRpl7 (4). (b) Immunoelectrophoresis of chick lens extracts. Electrophoresis was carried out in 1.5% Difco noble agar on a glass slide (2.5 X 7.5 cm) which was placed in the apparatus. Run for 2 h at 150 V in barbital buffer (pH 8.6, I’/2 = 0.05). Antiserum application and other details were described previously in Ref. (5).
43
a
b
8
2.
7
6. 5: tb
8. An assembly for preparative isoelectrofocusing and electrophoresis. (a) Schematic diagram of preparative column. a, upper buffer tray; b, lower buffer tray; 1, reservoir of elution buffer; 2, connecting tubing; 3, silicone rubber tubing, 4, stopper gel: 5, dialyzing tubing membrane; 6, porous plastic plate; 7. polyethylene tubing; 8, glass preparative column; 9, silicone rubber connector of upper tray; 10, peristalic pump; 11, test tube. l-9 were handmade. The column 8 was submerged in the lower tray buffer b which was cooled by circulating chilled water. (b) Photograph of (a). The large and small arrows indicate the column and magnetic stirring bar, respectively. FIG.
10
0.5
8
0.4
0.3
4
0.2
2
0.1
0
10
+
20
30
40 Fraction
50 NO.
60
70
80
100
x P 2
I 2 0
0 -
FIG.9. Preparative isoelectrofocusing in the apparatus. Discontinuous density gradient in the column (1 X 20 cm) made by 5-402 sucrose in which carrier ampholyte (pH 3 to 10) (LKB) 1% was contained. Upper tray contained 0.25 N NaOH. Lower tray contained 0.3% phosphoric acid. After electrofocusing for 12 h at 350 V under the ice-cooled condition, each fraction (7 drops) was collected through a guiding tube, 7 in Fig. 8, by siphon action (in this case, the pump 10 was not used). One milliliter of distilled water was added, then pH and absorbance of each fraction were measured. Electrofocusing samples were equine skeletal muscle myoglobin and methyl red.
ELECTROPHORESIS
AND ISOELECTROFOCUSING
ACKNOWLEDGMENTS I am grateful to Dr. A. Katoh, Mercy Hospital, Pittsburgh, Pennsylvania, for his critical reading of the manuscript and helpful suggestions. I am also indebted to Mrs. G. Takamatu, K. Koizumi, and Ms. C. Miwa of the Machine Shop for their kind help during the preparation of the manuscript.
REFERENCES 1. Mauer, H. R. (1971) Disc electrophoresis, de Gruyter, Berlin/New York.
APPARATUS
45
2. Gordon, A. H. (1972) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, E., eds.), Vol. 1, Part 1, North-Holland, Amsterdam/New York. 3. Catsimpoolas, N., and Drysdale, J. (eds.) (1977) Biological and Biomedical Applications of Isoelectric Focusing, Plenum, New York. 4. Struhl, K., Cameron, J. R., and Davis, R. W. (1976) Pmt. Nat. Acad. Sci. USA 73, 1471-1475. 5. Yoshida, K., and Katoh, A. (1971) Exp. Eye Rex 11, 122-131. 6. Yoshida, K. (1981) Pro&in, Nucleic Acid Enzyme [Tanpakushitrt Kakusan Kowo] 26, 258-260. [in Japanese]