JPH0358664B2 - - Google Patents

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, protein decomposition products,
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.

Particularly in the field of genetic engineering, which has recently attracted attention, autoradiography has been used to
Electrophoresis is essential for determining the base sequence of nucleic acids such as DNA. Electrophoresis operations for this purpose generally include operations in which a plurality of base-specific reactant mixtures of radioactively labeled DNA or DNA fragments are run in parallel along the direction of an electric field in an electrophoresis support medium. Then, after obtaining the multiple rows of migration patterns (collection of zones formed on the support medium by electrophoresis) as an autoradiograph, the zones in each row are compared with each other. The base sequence is determined. 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.

Paper, membrane filters, starch gel membranes, polyacrylamide gel membranes, etc. are generally used as support media for electrophoresis, and they are in the form of sheets with uniform thickness. Of these supporting media,
Gel films such as starch gel films and polyacrylamide gel films are formed by setting a support frame (spacer) around a flat support (support) made of an electrically non-conductive glass plate, organic polymer film, etc. It is formed by introducing a gel preparation solution onto a mold, sealing the top surface with another support if necessary, and then gelling it. Hereinafter, the entire structure in which a gel membrane is sandwiched between two supports may be simply referred to as an electrophoresis support medium.

However, when performing electrophoresis as described above using a rectangular sheet-like support medium of uniform thickness formed in this way, even if substances have the same molecular weight, their electrophoresis will be different. A phenomenon in which migration distance becomes non-uniform is likely to occur due to differences in migration positions in a direction perpendicular to the direction. 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 6. , the pattern tends to be such that the moving distance of the zone on both sides is shorter than the moving distance of the zone in the center. In addition, FIG. 6 shows an upper end starting point 62 on a support medium 61 formed on a support plate (support tool) 60.
Zones 63 and 63' obtained by electrophoresis from
This figure schematically shows the migration pattern of . This phenomenon is called the smiling effect, and is a major cause of a decrease in the accuracy of the information obtained, such as the base sequence, in operations that involve comparing and contrasting multiple electrophoresis columns, such as determining the base sequence of DNA. It is becoming. The main reason for the above-mentioned smiling effect is that the heat generated by the current passing through the support medium (Jule heat) is dissipated from the side edges, resulting in a temperature difference between the center and side surfaces. This can be mentioned. 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. Note that movement of Joule heat along the width direction occurs to compensate for these heat radiations, but even with regard to this heat movement, the heat radiation is likely to be compensated for in the central part of the support medium by the inflow of heat from both sides. In both side portions, heat only flows in from one side, resulting in insufficient compensation for heat radiation. For these reasons, the temperature of the side parts tends to be lower than the temperature of the central part, and as a result, the moving speed of charged substances is lower in the lower temperature side parts than in the central part, and the migration pattern is A smiling effect occurs.

For this reason, 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 that does not come into contact with the electrophoresis medium to suppress the generation of temperature differences. Efforts have been made to do so. Although a certain degree of effect can be obtained by doing this, it is difficult to increase the flatness of the metal plate, making it difficult to make the metal plate and support stick closely together over the entire surface, which tends to cause uneven heat radiation from the heat sink. 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 structure in which the buffer solution in the upper buffer solution tank 71 comes into contact with the entire back surface of the support plate 73 is also used.
In this device, the voltage applied between the upper electrode 72 and the lower electrode 76 is applied to the support medium 74 through the buffer solution in the upper buffer solution tank 71 and the lower buffer solution tank 75.
is applied to perform electrophoresis, but at this time, the upper buffer tank 71 that contacts the entire back surface of the support plate 73
The support plate 73 and the support medium 7 are
The temperature gradient in step 4 is reduced, and the occurrence of the smiling effect can be suppressed.

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 buffer 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 as high as 1500 to 3000V.
In addition, since the buffer solution 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 tends to occur.

For this reason, the present applicant has developed a structure in which a sheet-like support medium is sandwiched between flat support plates from both sides, and an upper buffer solution tank having an upper electrode inside and capable of containing and holding a buffer solution is attached to the support. A lower buffer solution tank having a lower electrode therein and capable of containing and holding a buffer inside the liquid is communicated with the lower end opening of the support plate;
Electrophoresis is carried out by transmitting the voltage applied between both electrodes to the support medium via both buffer solutions, and a heat-retaining water tank having a wall surface on at least one outer surface of the support plate is provided. An electrophoresis device is proposed in which temperature gradients in the support medium caused by water in the tank are prevented.

By using the above-mentioned device, the occurrence of the smiling effect can be prevented without causing the above-mentioned problems of cost and poor electrical insulation.

On the other hand, in this configuration, there are two buffer tanks.
Since one tank and one tank are used, the ease of attaching and removing each tank becomes a problem. In order to remove the support plate after electrophoresis is completed, it is first necessary to drain the water in the water tank and the buffer solution in each buffer tank. At this time, it is possible to turn the entire device upside down and discard the water and buffer solution, but in many cases a radioactive substance is added to the electrophoresed material in order to visually display the electrophoresis pattern.
In this case, there is a problem that the buffer solution in the lower buffer solution tank is contaminated with radioactive substances and cannot be thrown away as is, so it is preferable that each layer can be removed separately.

(Object of the Invention) In view of the above-mentioned circumstances, an object of the present invention is to provide an electrophoresis apparatus in which an upper buffer tank and the like can be easily attached and detached from a lower buffer tank.

(Structure of the Invention) The electrophoresis device of the present invention includes two substantially electrically non-conductive and non-flexible flat support plates that sandwich a support medium for electrophoresis, with a gap between the medium and the support plates. a support stand that can be fixed in a water-tight manner; an upper buffer tank that communicates with the upper end opening of the support plate and is capable of containing and holding a buffer; an upper buffer tank that communicates with the lower end opening of the support plate and has a buffer inside; A lower buffer tank capable of containing a liquid; upper and lower electrodes disposed in each of the upper and lower buffer tanks; An electrophoresis device capable of performing electrophoresis by applying a potential gradient in the vertical direction of the medium through a buffer solution contained and held in a solution tank, the support base for fixing the lower buffer solution tank. A pair of vertical plates extending upwardly provided on the top, a pair of side plates extending upward parallel to the vertical plates and holding the upper buffer tank; The present invention is characterized in that it is provided with an engaging means that can engage and disengage the plates, and that both plates can be engaged or disengaged simply by moving the side plate downward or upward relative to the vertical plate. That is.

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

FIG. 1 is a perspective view showing one example of a preferred embodiment of the electrophoresis device according to the present invention, FIG. 2 is a front view of this device as seen from the front, and FIG. FIG. 3 is a sectional view taken along the same line, 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, a heat-retaining water tank 5, and a lower buffer solution tank 7 are mounted on a support stand 1. In the upper buffer solution tank 2 and the lower buffer solution tank 7, an upper electrode 3 and a lower electrode 8 each made of a single platinum wire extending in the width direction are arranged, and a buffer solution is placed in each buffer solution tank 2, 7. When inserted, each electrode 3, 8 is adapted to be located in a buffer solution. These two electrodes 3 and 8 are connected to each buffer tank 2,
The external terminals 3a and 8a are connected to external terminals 3a and 8a which are attached to protrude from the side wall of 7.

The upper buffer tank 2 includes side plates 12, 13,
The box is surrounded by the rear lower surface plate 14 and the front plate 11 and has an open upper surface, and the front plate 11 has a notch 11b formed therein. The heat-retaining water tank 5 is a box that wraps around from the back side of the upper buffer solution tank 2 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. Ru. Here, as can be seen from the fact that the side plates 12, 13 and front plate 11 of the upper buffer solution tank 2 and the heat-retaining water tank 5 are common, the upper buffer solution tank 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.

The upper buffer tank 2 and the heat-retaining water tank 5 that are integrally configured in this way have side plates 12 and 13 that are connected to a pair of vertical plates 1 fixed to the top surface of the support base 1.
6 and 17 are engaged with the vertical plates 16 and 17 so as to sandwich them from the outside, and are held on the support base 1.
Side plates 12, 13 and vertical plates 16, 1
Specifically, the engagement in step 7 is performed as follows.
First engaging grooves 12a, 13a opening downward are formed at the lower ends of each side plates 12, 13, and second engaging grooves 16a, 17a opening upward are formed at the upper ends of each vertical plate 16, 17. At the same time, the inner surface of each side plate 12, 13 has a second groove that engages with the second engagement groove 16a, 17a as shown in the figure.
Engagement pins 12b, 13b are attached so as to protrude inward, and first engagement pins 16b, 17b that engage with first engagement grooves 12a, 13a are provided on the outer surface of each vertical plate 16, 17 as shown in the figure. installed. Therefore, by simply lowering the integrated upper buffer tank 2 and thermal water tank 5 from above with the side plates 12 and 13 sandwiching the vertical plates 16 and 176, the first engagement grooves 12a and 13a and the first The engagement pins 16b, 17b engage, and the second engagement grooves 12b, 13b and the second engagement pins 16a, 17a.
The upper buffer tank 2 and the heat-retaining water tank 5 can be attached to the support base 1 by engaging with each other.
In addition, the upper buffer tank 2 and the heat-retaining water tank 5 are supported by a stand 1.
When removing it, all you have to do is lift it upward, making it easy to attach and remove.

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 of substantially uniform thickness is held between support 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 20
The notch 11b on the front surface of the upper buffer solution tank 2 and the front opening 11a of the thermal water tank 5 are closed, but at this time, the buffer solution or water is not leaked 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. Assembly 2
0 is substantially electrically nonconductive and substantially inflexible, such as glass, ceramics, organic polymers (e.g., polymethyl methacrylate; polycarbonate of bisphenol A; cellulose acetate propionate, etc.) It is preferably transparent or white.

As shown in FIGS. 4A and 4B (a sectional view taken along the arrow B-B 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. thickness spacer 2
These spacers 23a, 23 sandwich 3a, 23b.
A gel conditioning solution is injected between two glass plates 21a and 21b surrounded by b to gel it, thereby forming a support medium 22 made of a sheet-like gel film with a uniform thickness. . Also, instead of this,
As shown in FIG. 5, spacers 23a and 23b of a predetermined thickness are sandwiched on the left and right sides of supports 24a and 24b made of two electrically non-conductive organic polymer films,
A gel film (support) 22 having a uniform thickness is sandwiched between two plastic films 24a and 24b surrounded by spacers 23a and 23b.
It may be held between two glass plates 21a and 21b.

As described above, the third
After the support assembly 20 is attached as shown by the chain line in the figure, a buffer solution is put into the upper and lower buffer solution tanks 2 and 7, and water is put into the heat-retaining water tank 5. After that, a predetermined voltage is applied between the external terminals 3a and 8a,
Perform electrophoresis. Note that the support assembly 20
The upper end of the glass plate 21 facing the front plate 11 is formed with a notch similar to the upper end of the front plate 11 among the glass plates 21a and 21b constituting the buffer solution tank 2. is in contact with the upper end of the gel membrane (support medium) 22 through this notch, and the lower end of the support assembly 20 protrudes into the lower buffer tank 7 so that the lower end of the gel membrane 22 is in contact with the upper end of the gel membrane (supporting medium) 22. It is designed to come into contact with the buffer solution in the buffer solution tank 7. Therefore, the voltage applied to the external terminals 3a and 8a acts on the gel membrane 22 through the buffer solution, and electrophoresis of the electrophoresed substances such as proteins and nucleic acids injected from the upper end of the gel membrane 22 is performed. .

In this case, the support assembly 20 is
Since the front opening 11a of the heat-retaining water tank 5 is watertightly closed, the water in the heat-retaining water tank 5 comes into contact with the support assembly 20 in this portion, thereby making the temperature of the support assembly 20 uniform. Therefore, the cooling (or heat-retaining) effect of the support medium 22 becomes almost uniform over the front surface, and it is possible to prevent the smiling effect from occurring.

Note that it is desirable to keep the water temperature in the heat-retaining water tank 5 substantially constant at all times, and as shown in the apparatus shown in FIGS.
It is preferable to provide

In order to remove the support assembly 20 after performing electrophoresis as described above, first, remove the support assembly 20 with the support assembly 20 attached to the front of the integrated upper buffer tank 2 and thermal water tank 5. is moved upward relative to the support base 1, and the first engagement grooves 12a,
13a and the first engagement pins 16b, 17b and the second engagement grooves 16a, 17a and the second engagement pin 12
b, 13b, and remove the upper buffer tank 2, thermal water tank 5, and support assembly 20 as one unit while leaving the lower buffer tank 7 fixed to the support stand 1 on the support stand 1. . The buffer solution in the upper buffer solution tank 2 and the water in the thermal water tank 5 are uncontaminated and can be thrown away as is, while the lower buffer solution is contaminated with radioactive substances added to the analyte material in the support medium. The buffer solution in the liquid tank 7 can be treated separately.

(Effects of the Invention) As explained above, according to the present invention, the upper buffer solution tank, the heat-retaining water tank, etc. can be connected to each other by simply moving the upper buffer solution tank, heat-retaining water tank, etc. up and down with respect to the support base that holds the lower buffer solution tank. Since the structure is configured so that they can be combined and separated, the buffer solution in the lower buffer tank contaminated with radioactive materials and the uncontaminated buffer solution in the upper buffer tank and the water in the thermal water tank can be separated. of,
It can be processed separately and the handling of the equipment is convenient.

[Brief explanation of drawings]

FIG. 1 is a perspective view showing one 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. 3 is a sectional view taken along arrow A-A in FIG. 2. , FIGS. 4A and 4B are sectional views showing a support assembly that is attached to the above-mentioned apparatus to perform electrophoresis, FIG. 5 is a sectional view showing different examples of the support assembly, and FIG. 6 is a conventional support assembly. FIG. 7 is a cross-sectional view showing an example of a conventional electrophoresis device. 1... Support stand, 2... Upper buffer tank, 3... Upper electrode, 5... Warm water tank, 7... Lower buffer tank,
8...Lower electrode, 9...Heater for water temperature adjustment, 1
2, 13... Side plate, 16, 17... Vertical plate, 20... Support assembly, 22...
gel membrane.

Claims (1)

[Scope of Claims] 1. Two substantially electrically non-conductive and non-flexible flat support plates that sandwich an electrophoresis support medium,
a support base that can be fixed to maintain watertightness between the medium and the support plate; an upper buffer tank communicating with the upper end opening of the support plate and capable of containing and holding a buffer solution therein; and a lower end opening of the support plate. a lower buffer tank capable of containing a buffer solution therein; and upper and lower electrodes disposed in each of the upper and lower buffer tanks, and a predetermined voltage is applied between the upper and lower electrodes. An electrophoresis apparatus capable of performing electrophoresis by applying a potential gradient in the vertical direction of the medium through buffer solutions contained and held in the upper and lower buffer solution tanks, wherein the lower buffer solution a pair of vertical plates extending upwardly on a support base for fixing the tank; a pair of side plates extending upward parallel to the vertical plates and holding the upper buffer tank; and both plates. and an engagement means capable of engaging and disengaging both plates by relative vertical movement of the plate, and engaging or disengaging the plates simply by moving the side plate downward or upward relative to the vertical plate. An electrophoresis device characterized by being able to perform 2. The electrophoresis device according to claim 1, wherein the side plate and the vertical plate engaged by the engagement means constitute means for fixing the support plate to a support base. 3. The electrophoresis device according to claim 1, wherein the support plate is a glass plate.