JPS61278752A - Electrophoretic instrument - Google Patents

Abstract

PURPOSE:To maintain uniformly the temp. of a supporting medium and to prevent the generation of a smiling effect by bringing the water in a heat retaining water tank into contact with the outside surface of a supporting plate and disposing heaters for adjusting water temp. into the heat retaining water tank. CONSTITUTION:This electrophoretic instrument is basically constituted by installing an upper buffer soln. tank 2, a heat retaining water tank 5 and a lower buffer soln. tank 7 onto a supporting base 1. A supporting assembly 20 is attached to the front surface of the instrument and thereafter the buffer soln. is put into the upper and lower buffer soln. tanks 2, 7. Water is put into the tank 5.The assembly 20 closes watertightly the aperture 11a at the front surface of the tank 5 and therefore the water in the tank 5 contacts with the assembly 20 in this part so that the temp. thereof is made uniform. The generation of a difference in the water temp. between the central part and both right and left ends is obviated by the effect of the heaters 9, 10 for adjusting water temp. provided at the right and left side ends in the tank 5 and the water temp. in the tank 5 is maintained uniform and constant over the entire part.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of the Invention) The present invention is useful for separating and analyzing substances that have ionizing groups in solution, such as proteins and nucleic acids, based on the differences in charge and molecular weight of their particles. The present invention relates to the electrophoresis device used.

(Background of the Invention) Proteins and protein decomposition products are produced in a sheet-like support medium such as a gel membrane or m-paper impregnated with a buffer solution.

Electrophoresis operations have been known for some time, and their main purpose is to separate charged molecules or particles, such as nucleic acids and nucleic acid decomposition products, by utilizing the phenomenon in which they move under the influence of an electric field. It is used for the separation and fixation of biopolymer substances such as.

Especially in the field of genetic engineering, which has recently attracted attention,
In order to determine the base sequence of a nucleic acid such as DNA using autoradiography, it is essential to perform an electrophoretic operation. Electrophoretic procedures used for this purpose are generally
This involves an operation in which a plurality of base-specific reaction mixtures of radioactively labeled DNA or DNA fragments are migrated in parallel along the direction of an electric field in a support medium for electrophoresis. Then, a multi-row migration pattern (a collection of zones formed on the support medium by electrophoresis) is obtained by the migration.
After obtaining an autoradiograph, the base sequence is determined by comparing the zones in each row with each other. In other words, the comparison is made using the principle of electrophoresis that base-specific reactants having the same molecular weight migrate to the same position if the starting point of electrophoresis is the same.

Generally, paper, membrane filters, starch gel membranes, polyacrylamide gel membranes, etc. are used as support media for electrophoresis, and they are in the form of sheets of uniform thickness.

Among these supporting media, gel membranes such as starch gel membranes and polyacrylamide gel membranes are supported by electrically non-conductive glass plates,
The gel preparation solution is introduced onto a mold formed by setting a support frame (spacer) around a flat support (support) made of an organic polymer film, etc., and if necessary, cover the top surface with another support. It is formed by sealing it and then gelling it. Hereinafter, the gel membrane sandwiched between two supports may also be simply referred to as an electrophoresis support medium.

However, when the above electrophoresis operation is performed using the rectangular sheet-like support medium of uniform thickness formed in this way, even if substances have the same molecular weight, their migration positions differ. Due to the difference in the electrophoresis distance, the phenomenon of uneven migration tends to occur. In other words, the moving speed of charged substances (i.e., the moving distance of the zone) generally tends to be smaller on both sides than in the center, and therefore the migration pattern after a certain period of time is as illustrated in Figure 7. , the moving distance of the zone on both sides is L in the center
13 [The pattern is likely to be shorter than the travel distance of the noru zone. Note that FIG. 7 schematically shows the electrophoresis pattern of zone 73.73= obtained by electrophoresis from the upper end starting point 72 on the support medium 71 formed on the support plate (supporter>70). This phenomenon is called the smiling effect, and in operations that involve comparing and contrasting multiple electrophoresis columns, such as determining the base sequence of DNA, etc., the accuracy of the information obtained, such as the base sequence, decreases. The main cause of the above-mentioned smiling effect is that the heat (Joule heat) generated by the passage of current through the support medium is dissipated from the side edges, and the central part In other words, the generated Joule heat is radiated only from the front and back surfaces in the central portion of the support medium, but in the side portions, heat is further radiated from the side edges. A3: To compensate for these heat dissipations, Joule heat also moves along the width direction, but in the center of the support medium, the heat dissipation is easily compensated for by the inflow of heat from both sides. In the case of both side surfaces, heat inflows only from one side, resulting in insufficient compensation for heat radiation.For these reasons,
The temperature at the side portions tends to be lower than that at the center, and as a result, the moving speed of charged substances at the lower temperature side portions is lower than that at the center, creating a smiling effect on the migration pattern. ◎ Therefore, in order to prevent the smiling effect from occurring, a heat dissipation plate made of a metal plate with good thermal conductivity is attached to the surface of the support plate on the side not in contact with the electrophoresis medium to create a temperature difference. Efforts are being made to suppress this. Although some effect can be obtained by doing this, it is difficult to increase the flatness of the metal plate, so it is difficult to make the metal plate and support stick closely together over the entire surface, and the heat radiation of the heat sink is likely to be uneven. Due to this problem, it is difficult to sufficiently prevent the smiling effect, and the work of attaching a heat sink is also complicated.

For this reason, as shown in FIG. A device in which the liquid comes into contact with the entire back surface of the support plate 83 is also used.

In this way, the voltage applied between the upper electrode 82 and the lower electrode 86 can be applied to the upper buffer tank 81 and the lower Mm
applied to the support medium 84 via the buffer solution in the liquid tank 85;
At this time, the upper buffer tank 8 contacts the entire back surface of the support plate 83.
The buffer solution in 1 reduces the temperature gradient of the support plate 83 and the support medium 84, making it possible to suppress the occurrence of the smiling effect.

However, in this case, a large amount of relatively expensive buffer solution is required in order to prevent a temperature difference from occurring in the 1ili solution, so there is a problem in that the expense for performing electrophoresis becomes high. Furthermore, the voltage applied between the upper and lower electrodes for electrophoresis is usually 1500 to 300
Since the voltage is as high as 00V and the 11m liquid has good conductivity, if the upper buffer solution tank is extended downward and brought closer to the lower buffer solution tank, the problem of poor electrical insulation between the two is likely to occur.

Further, even though the temperature gradient can be reduced by using the buffer solution, the heat dissipation from the side portions of the buffer solution is greater than the heat dissipation from the center portion, and the occurrence of the smiling effect to some extent is unavoidable.

(Object of the Invention) In view of these problems, the present invention is capable of suppressing the smiling effect at a relatively low cost without requiring a large amount of buffer solution, and in addition, it is possible to suppress the smiling effect at a relatively low cost without requiring a large amount of buffer solution. It is an object of the present invention to provide an electrophoresis device that can be easily electrically insulated.

(Structure of the Invention) The electrophoresis device of the present invention includes a substantially electrically non-conductive and non-flexible flat support plate that holds a sheet-like electrophoresis support from both sides and is fixed in place. a support base capable of watertightly fixing the support plate, and communicating with the upper end opening of the support plate;
an upper buffer solution tank capable of containing and holding a buffer solution therein, a lower buffer solution tank communicating with the lower end opening of the support plate and capable of containing and holding a buffer solution therein, and each of the upper and lower buffer solution tanks. The upper and lower electrodes are arranged, and by applying a predetermined voltage between the upper and lower electrodes, the support medium is moved in the vertical direction through the buffer solution contained and held in the upper and lower buffer solution tanks. An electrophoresis device is configured to perform electrophoresis by applying a potential gradient to the electrophoresis device, and the electrophoresis device is provided with a heat-retaining water tank whose wall is at least one outer surface of the support plate,
Water temperature adjusting heaters extending in the direction of the electric field applied to the support medium are arranged along both sides of the support plate in this heat-retaining water tank.
It is characterized in that the temperature of the support medium is made uniform by the water whose temperature has been made uniform by the heater, thereby preventing the occurrence of temperature gradients.

(Embodiments) Preferred embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a perspective view showing a preferred embodiment of the electrophoresis device according to the present invention [Q-type], FIG. 2 is a front view of this device as seen from the front, and FIG. 3 is an arrow A-- 3 is a sectional view taken along line A, and the present device will be described below using FIGS. 1 to 3 together.

The basic configuration of this device is that an upper buffer solution tank 2, warm water (n5) and a lower buffer solution tank 7 are mounted on a support stand 1. An upper electrode 3 and a lower electrode 8 made of a single platinum wire extending in the direction are arranged, and when a buffer solution is placed in each of the slow liquid tanks 2 and 7, each electrode 3.8 is placed in the buffer solution. These two electrodes 3.8 are located in each buffer tank 2, respectively.
External terminal 3a protruding and attached to the outside of the side wall of ゜7
, connected to 8a.

The upper W1116itj tank 2 has side panels 1/-to 12, 13.
, a box whose upper surface is open and surrounded by the rear lower surface plate 14 and the front plate T1, and the front plate 11 has a notch 11b formed therein. The warm water PI 35 is a box that flows around from the rear side of the upper buffer solution 12 to the bottom side and extends downward to the vicinity of the lower buffer solution tank 7, and is formed of side plates 12, 13, a back plate 15, and a front plate 11. . Here, as can be seen from the fact that the upper wJ buffer solution tank and the side plates 12.13 and front plate 11 of the warm water 1115 are common, the upper buffer solution [2
and the heat-retaining water tank 5 are integrally constructed. Note that a front opening 11a is formed at the front of the heat-retaining water tank 5.

In addition, a pair of left and right water temperature adjustment heaters 9.10 extending vertically are arranged near the left and right ends of the heat-retaining water tank 5.
The heaters 9 and 10 can prevent a difference in water from occurring between the center and the side edges in the heat-retaining water tank 5.

The upper buffer solution tank 2 and the heat-retaining water tank 5, which are integrally constructed in this way, are constructed such that each side plate 12.13 sandwiches a pair of vertical plates 16.17 fixedly installed on the upper surface of the support base 1 from the outside. It engages the vertical plates 16, 17 and is held on the support base 1.

Specifically, the engagement between the side plates 12, 13 and the vertical plates 16, 17 is carried out as follows.

First engaging grooves 12a and 13a opening downward are formed at the lower end of each side plate 12.13, and each vertical plate 1
6. M2 engagement groove 16a opened upward at the upper end of 17,
17a is formed and each side plate 12.1
Second engagement pins 12b and 13b that engage with second engagement grooves 16a and 17a are attached to the inner surfaces of the vertical plates 16 and 17 as shown in the figure, protruding inward, and the outer surfaces of each vertical plate 16 and 17 are provided with second engagement pins 12b and 13b that engage with the second engagement grooves 16a and 17a, respectively, as shown in the figure. First engaging pins 161) and 17t) that engage with the first engaging grooves 12a and 13a are attached as shown in FIG.

For this reason, the side plates 12.13 and the vertical plates 16
．． By simply lowering the integrated upper buffer solution tank 2 and heat-retaining water tank 5 from above with the upper buffer tank 176 sandwiched therebetween, the first engagement grooves 12a, 13a and the first engagement bins 161), 17b are engaged with each other. , second engagement grooves 12b, 13b and second engagement pin 1
6a and 17a are engaged to form an integrated upper buffer tank 2
and a heat-retaining water tank 5 can be attached to the support stand 1.

Furthermore, when removing the upper buffer solution tank 2 and the warm water 1fF5 from the support stand 1, it is sufficient to simply lift them upward, making it easy to attach and remove them.

The lower buffer tank 7 is attached to the support stand 1 and held so that it can be easily removed.As shown by the two-dot chain line in FIG. After attaching the support assembly 20 in which a sheet-like support medium having a substantially uniform thickness is held between support plates made of glass plates,
The upper buffer solution tank 7 and the heat-retaining water tank 5 are attached to the support base 1 as described above. In this way, the support assembly 2
0 closes the front notch 11b of the upper buffer solution tank 2 and the front opening 11a of the insulating water [5], but at this time, the buffer solution or water leaks from between the contact surface of the support assembly 20 and the front plate 11. A buffer tank gasket 4 and a water tank gasket 6 are provided on the front plate 11 so that the water tank gas is not removed. Assembly 20 is substantially electrically non-conductive and substantially inflexible, such as glass, ceramics, organic polymers (e.g., polymethyl methacrylate; polycarbonate to bisphenol: cellulose acetate brobionate, etc.). It is preferably transparent or white.

As shown in FIGS. 4A and 4B (a sectional view taken along arrow B-8 in FIG. 4A), the support assembly 20 is mounted on the left and right sides of two glass plates 21a and 21b serving as support plates. Thick spacer 23a.

231) and between the two glass plates 21a and 21b surrounded by the spaces 1+23a- and 23b, a gel conditioning solution is injected between the two glass plates 21a and 21b to gel it, resulting in a sheet-like gel film with a uniform thickness. A support medium 22 is used.

Alternatively, as shown in FIG. 5, supports 24a, 2 made of two electrically non-conductive organic polymer films may
Spacers 23a and 23b of a predetermined thickness are sandwiched on the left and right sides of 4b, and a gel film (supporting medium) 22 of uniform thickness is sandwiched between two plastic films 24a and 24b surrounded by the spacers 23a and 23b. An object may be held between two glass plates 21a and 21b.

After attaching the support assembly 2o to the front of the electrophoresis apparatus as shown by the chain line in FIG. Put in. Thereafter, a predetermined voltage is applied between the external terminals 3a and 8a to perform electrophoresis. Note that, among the glass plates 21a and 21b constituting the support assembly 2o, the upper end of the glass plate 21 facing the front plate 11 is formed with a cutout similar to the notch at the upper end of the front plate 11, and an upper buffer The buffer solution in the liquid tank 2 comes into contact with the upper end of the gel family (supporting medium) 22 through this notch, and the lower end of the support assembly 20 protrudes into the lower buffer solution tank 7. The lower end of the gel membrane 22 is inside the lower buffer tank 7! 1. It is designed to come into contact with a buffer solution. Therefore, the voltage applied to the external terminals 3a and 8a acts on the gel II 22 through the gm liquid, and the electrophoresed substances such as proteins and nucleic acids injected from the upper end of the gel membrane 22 are electrophoresed. .

In this case, since the support assembly 2o watertightly closes the front opening 11a of the heat-retaining water tank 5, the water in the heat-retaining water WI5 flows into the support assembly 20 in this part.
, which equalizes the temperature of the support assembly 20. At this time, the water temperature adjustment heaters 9.10 provided at the left and right ends of the heat retention water tank 5 are used to prevent any difference in water temperature between the center and the left and right sides, and the entire hot water in the heat retention water 1f15 is heated. It can be kept uniform and constant at all times, so that the cooling (or heat retention) effect of the support medium 22 is almost uniform over the front surface, and the smiling effect can be prevented from occurring.

(Effects of the Invention) As explained above, according to the present invention, the water in the heat-retaining water tank is applied to the outer surface of the flat support plate that sandwiches the support medium to which a voltage is applied between the upper and lower atVE liquid tanks. In addition, water temperature adjustment heaters extending in the direction of the electric field applied to the support medium are installed near both side edges of the water tank, so that the water in the water tank is heated This lagoon degree can be maintained evenly by
The temperature of the surface of the support can be maintained substantially uniform by using water, and the temperature of the support medium is also made substantially uniform, making it possible to prevent the smiling effect from occurring during electrophoresis.

Furthermore, in the case of the present invention, the insulating water bath may be filled with ordinary water, and the amount of buffer solution placed in the upper and lower buffer baths is the minimum amount necessary to transfer the voltage of each electrode to the supporting medium. Since silk can be used, costs can be reduced.

In addition, the upper Ill liquid tank and the lower m* liquid tank can be spaced apart from each other, making it easy to insulate both types of liquids.

Embodiments of the present invention will be described below with reference to Examples, but the content of the present invention is not limited thereto.

Example and Comparative Example Two glass plates (0.5X20X40a), however,
One glass plate has a notch for communication with the upper buffer tank 2.
A spacer of 0 μm was provided and the glass plate was fixed with a clip. Separately, an acrylamide aqueous solution having the following composition was prepared.

Acrylamide 11.87gN, N'-methylenebisacrylamide 0.639 Urea 42. (
1 Tris(hydroxymethyl)aminomethane [CAS
Reuistry NQ77-86-1,,08
g boric acid 0.
55 gEDTA-2Na salt 0.0939 Add water to make 100d.

To the aqueous solution were added 1.3 d (5% by weight) of ammonium beroxonisulfate and 33 µm of N, N, N', N'-tetramethylethylenediamine as a polymerization initiator, and the mixture was poured between the glass plates to form a polyacrylamide gel film. Ta. Before the gel hardens, add a layer of 4.
An insert for forming a slot for 00μ milk was inserted.

A DNA sample for electrophoresis was prepared as follows.

Using TAK A RA DNA sequence kit,
According to the instructions, a T reaction was performed using M-13108 and [α-Pcod CTP (manufactured by Ama Jam Japan) to prepare P-labeled DNA.

Electrophoresis support medium (polyacrylamide gel membrane) as described above
Two glass plates having the same structure were fixed in such a way that the cutout portions matched with the upper SL impact-resistant liquid cutouts 11b. Next, fill water into the heat-retaining water tank 5, and then fill the upper and lower parts!
11 liquid bath buffer was added.

Water temperature adjustment heaters 9 and 10 (30W cartridge heaters) disposed along both sides of the heat-retaining water tank 5 were energized to keep the water temperature in the heat-retaining water tank 5 uniform and constant. Electrophoresis was performed using all the slots with P-labeled DNA using the upper buffer tank 5 as a cathode and the lower buffer tank 7 as an anode. After applying a voltage of 1500 V and performing electrophoresis for about 3 hours, the glass plate was removed and the gel film was transferred to paper. The gel film was then covered with Zalan wrap and heated to -7
Results were obtained by performing autoradiography for 15 hours at 0°C. The migration pattern after electrophoresis showed a nearly straight line as shown in FIG. 6, and the occurrence of the smiling effect was negligible.

Further, as a comparative experiment, electrophoresis was conducted in the same manner without adding water to the heat-retaining water tank 5. A smiling effect appears in the migration pattern after electrophoresis as shown in Figure 7, and the migration distance in the center and on both sides is about 3.
There was a difference of 1.

[Brief explanation of the drawing]

FIG. 1 is a perspective view showing an example of an electrophoresis device according to the present invention, FIG. 2 is a front view of the device seen from the front, and FIG.
Cross-sectional view along arrow A-A in the figure, Figures 4A and 4B
The figure is a sectional view showing a support assembly that is attached to the above device to perform electrophoresis, Figure 5 is a sectional view showing different examples of the support assembly, and Figure 6 is an example of electrophoresis using the electrophoresis apparatus of the present invention. , a schematic diagram showing a state in which the smiling effect of an electrophoretic pattern developed on a support medium is prevented from occurring; FIG. FIG. 8 is a cross-sectional view showing an example of a conventional electrophoresis device. 1... Support stand 2... Upper buffer tank 3...
・Upper electrode 5...Heat water tank 7...Lower buffer tank 8...Lower electrode 9.10.
...Water temperature adjustment heater 12.13...Side plate 16.17...Vertical plate 20...Support assembly 22...Gel film Fig. 4B Fig. 6 Fig. 7 Fig. 8

Claims (1)

[Scope of Claims] 1) A substantially electrically non-conductive and non-flexible flat support plate that sandwiches a sheet-shaped support medium for electrophoresis from both sides is placed and fixed in a fixed position, and the support plate a support base capable of watertightly fixing the support plate, an upper buffer solution tank communicating with the upper end opening of the support plate and capable of containing and holding a buffer solution therein, and an upper buffer tank communicating with the lower end opening of the support plate containing the buffer solution therein. a lower buffer tank capable of containing and holding;
and upper and lower electrodes disposed in each of the upper and lower buffer tanks, and is housed and held in the upper and lower buffer tanks by applying a predetermined voltage between the upper and lower electrodes. In an electrophoresis device in which electrophoresis is performed by applying a potential gradient in the vertical direction of the support medium via a buffer solution, a portion of the outer surface of at least one of the support plates that clamps the support medium is a wall surface. A heat-retaining water tank is formed, and in the heat-retaining water tank, heaters for water temperature adjustment are disposed at side ends extending in the direction of the electric field applied to the support medium along both side edges of the support plate. Characteristic electrophoresis device. 2) The electrophoresis device according to claim 1, wherein the flat support plate is a substantially electrically non-conductive and non-flexible glass plate, ceramic plate or organic polymer plate. .