JPH0551103B2 - - Google Patents

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention is directed to the separation and analysis of substances having ionizing groups in solution, such as proteins and nucleic acids, based on the differences in charge and molecular weight of their particles. This relates to an electrophoretic sheet used for.

(Prior art) Charged molecules or particles such as proteins, protein decomposition products, nucleic acids, and nucleic acid decomposition products move under the influence of an electric field in a sheet-like medium such as a gel membrane or filter paper impregnated with a buffer solution. Electrophoresis operations have been known for separating these substances by utilizing the phenomenon of electrophoresis, and are particularly used for the separation and immobilization of biopolymer substances as described above.

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 the electric field of an electrophoresis medium. and,
After obtaining the multiple rows of migration patterns (collection of zones formed on the medium by electrophoresis) as autorandom, the base sequence is determined by comparing the zones in each row with each other. will be carried out. That is, the comparison is made using the principle of electrophoresis that base-specific reactants having the same molecular weight move to the same position if the starting point of electrophoresis is the same.

In general, filter paper, membrane filters, starch gel membranes, polyacrylamide gel membranes, etc. are used as electrophoresis media, and they are in the form of sheets with uniform thickness. When using gel films such as starch gel films and polyacrylamide gel films as these media, conventionally a support frame (spacer) is installed around a flat support (support) made of a non-conductive material such as a glass plate. The gel preparation solution formed in the mold was introduced, and if necessary, the upper surface was further sealed with another support, and then the mold was gelled.
However, this gel film formation method is very troublesome;
This required the experimenter to perform cumbersome work before performing electrophoresis operations.

In view of the above-mentioned circumstances, an electrophoresis sheet that can be easily used without the need for troublesome operations such as gel preparation has been proposed (for example, JP-A-59-126237).
This electrophoretic sheet consists of two sheet materials made of non-conductive organic polymer films that are placed facing each other with a spacer of a predetermined thickness sandwiched between the left and right ends, and a gel film for electrophoresis is uniformly spread between these sheet materials. It is made by sandwiching it to a certain thickness. In the case of this electrophoretic sheet, it can be sold with a gel membrane arranged between two polymer films as described above.
Electrophoresis experimenters can purchase this electrophoresis sheet and easily perform electrophoresis operations.

By the way, in the above electrophoretic sheet, a non-electrophoretic substance is injected and held at the upper end of the gel membrane as an electrophoretic medium (that is, the end that does not face the spacer and is located on the upper side during electrophoresis operation). A slot is provided for the These slots are shaped to extend from the upper end of the gel membrane toward the center, and are usually arranged in plural in the left-right direction of the gel membrane. As mentioned above, when forming a gel film by pouring a gel preparation between flat supports such as glass plates, before pouring the gel preparation, between both supports,
Usually, a tooth-shaped member called a sample comb is arranged, and after the preparation solution has gelled, the member is pulled out to form the above-mentioned slot. However, in manufacturing the electrophoretic sheet as described above, since a web-like gel film that has already been gelled is held between the sheet materials, the slots are formed by punching this web-like gel film.

(Problem to be solved by the invention) When performing electrophoresis using the electrophoresis sheet created in this way, the electrophoresis sheet is held between plate glasses, etc., and then set in an electrophoresis apparatus. . Next, the slot of the electrophoresis sheet is cleaned with a cleaning solution using a syringe, etc.
Thereafter, the substance to be electrophoresed is injected into this slot using a microsyringe or the like. However, in this case, the thickness of the gel membrane, which is the medium for electrophoresis, is thin, and the diameter of the needle of the injection needle or microsyringe is larger than this thickness. There is a problem in that it is difficult to insert a hypodermic needle or a microsyringe needle. Specifically, as shown in FIG. 8, for example, the thickness of the gel film 103 is 200 μm, while the diameter of the needle 105 of the microsyringe is 250 μm. hold 2
In order to insert the polymer films 101 and 102 between the polymer films 101 and 102,
2 has to be pushed apart and inserted, which makes it difficult to insert. In particular, the upper end 1 of the polymer film 102
If 02a is curled inward as shown in the figure, its insertion becomes even more difficult.

(Means for solving the problem) Therefore, in the present invention, when cleaning the slot and injecting the substance to be electrophoresed into the slot, the needle of the injection needle or the microsyringe is attached to two polymer films. It is an object of the present invention to provide an electrophoretic sheet that can be easily inserted into the slots held between the sheets, thereby making it easy to clean the slots and inject a substance to be electrophoresed into the slots, and as a means for this purpose, The electrophoretic sheet of the present invention is constructed as follows.

That is, in this electrophoretic sheet, gel spacers of a predetermined thickness are sandwiched between the left and right ends of two supports made of conductive organic polymer films, and the two polymer films are separated between the gel spacers. In an electrophoretic gel sheet in which an electrophoretic medium (gel membrane) is sandwiched between the electrophoretic medium (gel membrane) with a uniform thickness, there is a gap between two polymer films at the upper end of the gel sheet that is thicker than the thickness of the electrophoretic medium. The spacer is used to widen the gap between the polymer films at the upper end of the gel sheet.

(Function) By configuring the electrophoresis sheet as described above, the interval between the two polymer films at the upper end of the gel sheet is widened by the interval expansion spacer, so that an injection needle, a microsyringe needle, etc. can be inserted between the two polymer films. This makes it easier to clean the upper end of the gel membrane held between the polymer films and to supply the electrophoresed substance to this upper end.

(Embodiments) Hereinafter, preferred embodiments of the present invention will be described based on the drawings.

FIG. 1 is a perspective view showing an electrophoresis device using a gel sheet for electrophoresis according to the present invention, and this electrophoresis device basically consists of a support base 1, a lower buffer tank 2, a pair of supports 3, 3, an upper buffer tank 4 and an electrophoresis sheet assembly 10. The lower buffer tank 2 is fixed on the support base 1, and the columns 3, 3 are also fixed on the support base 1. An upper buffer tank 4 is attached to the upper portions of the columns 3, 3 by set screws 3a, 3a (only one screw is shown). Furthermore, the electrophoretic sheet assembly 10 includes:
The upper end is attached to the upper buffer solution tank 4 so as to face the front notch of the upper buffer solution tank 4, and the lower end is attached so as to enter into the lower buffer solution tank 2. At this time, the electrophoretic sheet assembly 10 is attached using the clips 9. Note that upper and lower electrodes 5 and 6 (the lower electrode 6 is not shown) each made of one platinum wire are arranged in the upper and lower buffer solution tanks 2 and 4, and these electrodes 5,
6 are connected to external terminals 5a, 6a protrudingly attached to the side wall members of each buffer solution tank 2, 4, respectively. For this reason, the upper and lower buffer tanks 2, 4
Electrophoresis can be performed by adding a buffer solution to the buffer and applying a predetermined voltage to external terminals 5a and 6a.

Next, the configuration of the electrophoretic sheet assembly 10 will be described in detail. As shown in the perspective view of FIG.
1 and the flat glass plate 12, the gel sheet 20
Holding it in place. Note that the chipped glass plate 11 and the flat glass plate 12 may be made of ceramic plates instead of glass plates.

On the other hand, the gel sheet 20 is composed of two sheet materials 21 made of a non-conductive organic polymer film, as shown in FIG. , 22 are arranged facing each other with gel spacers 23a, 33b of a predetermined thickness sandwiched between them on the left and right sides, and gel spacers 23a, 22 are placed between both sheet materials 21, 22.
It is formed by sandwiching an electrophoretic gel membrane 24 having the same thickness as 23b. At this time, the chipped glass plate 1
A cutout 21a having the same shape as the cutout formed at the upper end of the chipped glass plate 11 is formed at the upper end of the sheet material 21 on the side facing the chipped glass plate 11, and a plurality of cutouts 21a for sample injection are formed at the upper end of the gel film 24. A slot 24a is formed.

The gel sheet 20 having such a configuration is made as follows. First, acrylamide, BIS, agarose, tris(hydroxymethyl)aminomethane, boric acid, and EDTA.
A polyacrylamide gel film 24 is obtained by adding ammonium peroxonisulfate and TEMED as a polymerization initiator to an aqueous solution of 2Na salt to form a layer with a thickness of 0.2 mm. Next, a part of this gel film 24 is punched out into an appropriate shape and removed to form a slot 24a, and then the gel film 24 is covered with another sheet material 22 (cover sheet) made of polymer film. Then, the electrophoretic gel sheet 20 having the above configuration is produced.

Furthermore, this slot 2 is located above the gel film 24.
A spacer 25 for widening the gap having a notch 25a facing 4a is disposed, and the lower end of this spacer 25 is held between two sheet materials 21 and 22 as shown in FIG. has been done. The thickness of the spacer 25 is greater than the thickness of the gel film 24, so the upper end of the sheet material 21 is pushed outward by the spacer 25 as shown. Notch 25a formed in spacer 25
As a result, the slot 24 communicates with the outside, so insert the needle 31 of the microsyringe 30 through this communication part as shown in FIG.
4 and supply the substance to be electrophoresed into the slot 24. in this case,
The spacer 25 may be manufactured by being attached to the sheet material and integrated with the gel sheet at the time of manufacturing the gel sheet, or it may be attached (inserted) to the gel sheet before electrophoresis (before the gel sheet is set in the electrophoresis apparatus).
It's okay. Furthermore, the material of the spacer 25 is as follows:
It is preferable to use polyethylene, polyvinyl chloride, polyester, etc.

To perform electrophoresis using the electrophoresis apparatus configured as described above, a buffer solution is poured into the upper and lower buffer solution tanks 2 and 4, and a syringe or the like is inserted into the slot 24a at the upper end of the gel membrane 24 of the gel sheet 20. Send the buffer solution as a washing solution to this slot 2.
Clean the inside of 4a. After this, this slot 24a
A predetermined sample liquid may be injected into the chamber using a microsyringe or the like, and then a predetermined voltage may be applied between external terminals 5a and 6a connected to the upper and lower electrodes 5 and 6. At this time, the interval between the two sheet materials 21 and 22 sandwiching the slot 24a is expanded by the interval expanding spacer 25, so that it is easy to insert a hypodermic needle, a microsyringe needle, etc., and the work is easy. . The buffer in the upper buffer tank 4 comes into contact with the upper end of the gel membrane 24 through the notch 11a of the chipped glass plate 11 and the notch 21a formed in the sheet material 21 of the gel sheet 20, and the buffer in the lower buffer tank 4 Since the lower end of the electrophoretic sheet assembly 10 protrudes into the lower buffer solution tank 2, the buffer solution in the gel membrane 24 also comes into contact with the lower end of the gel membrane 24. Therefore, the voltage applied to the external terminals 5a, 6a acts on the gel membrane 24 through the buffer solution, allowing the sample injected from the slot 24a to undergo electrophoresis within the gel membrane 24.

Incidentally, the spacer for expanding the gap has the notches 25 facing each slot 24a of the gel film 24 as described above.
Of course, a large cut 25b facing each other may be formed for each of the plurality of slots 24a, as shown in FIG. 7.

(Effects of the Invention) As explained above, according to the present invention, a spacer for increasing the gap, which is thicker than the thickness of the electrophoresis medium, is sandwiched between two polymer films at the upper end of the gel sheet, and the polymer film at the upper end of the gel sheet is Since the distance between the films is widened, it is easy to insert a syringe needle, a microsyringe needle, etc. between the films, and it is easy to clean the upper end of the gel membrane held between the polymer films and perform electrophoresis on this upper end. It is easy to perform work for supplying materials.

[Brief explanation of the drawing]

FIG. 1 is a perspective view showing an example of an electrophoresis device using the gel sheet for electrophoresis according to the present invention, FIG. 2 is a perspective view showing an electrophoresis sheet assembly used in the device shown in FIG. 1, and FIG. , arrow in Fig. 2 -
4 is a perspective view showing the gel sheet for electrophoresis according to the present invention exploded into component parts, and FIG. 5 is a sectional view taken along the arrow - in FIG. 4. , FIG. 6 is a front view of the above gel sheet for electrophoresis, FIG. 7 is a front view showing different examples of the gel sheet for electrophoresis according to the present invention, and FIG. 8 is a sectional view showing a conventional gel sheet for electrophoresis. . DESCRIPTION OF SYMBOLS 1... Support stand, 2... Lower buffer tank, 4... Upper buffer tank, 8... Warm water tank, 10... Electrophoresis sheet assembly, 11... Chipped glass plate, 12
... Flat glass plate, 20 ... Gel sheet, 21,2
2... Sheet material, 23a, 23b... Gel spacer, 24... Gel film, 24a... Slot, 2
5... Spacer for expanding the interval, 25a, 25b...
Depth of cut, 30...Micro syringe.

Claims (1)

[Claims] 1. Gel spacers of a predetermined thickness are sandwiched between the left and right ends of two supports made of non-conductive organic polymer films, and between the two polymer films between these gel spacers. In an electrophoretic gel sheet in which an electrophoretic medium is sandwiched between the electrophoretic medium to a uniform thickness, a spacer for expanding the distance is provided between the two polymer films at the upper end of the gel sheet, which is thicker than the thickness of the electrophoretic medium. A gel sheet for electrophoresis, characterized in that the two polymer films are sandwiched together to widen the gap between the two polymer films at the upper end of the gel sheet. 2. A plurality of slots for injecting a substance to be electrophoresed are formed in the upper end of the electrophoresis medium in a row in the left-right direction, and the expansion spacer has a notch that communicates the upper part of the slot with the outside. A gel sheet for electrophoresis according to claim 1.