JP5287569B2 - Method for producing gel cassette for electrophoresis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing a gel cassette for electrophoresis capable of forming a gel interface horizontally with excellent reproducibility. <P>SOLUTION: An electrophoretic gel cassette substrate (1a) and a gel cassette substrate (1b) for electrophoresis are integrated through a spacer (1c). The lower end is sealed with an end face sealing tape (3), and a shielding structure (4) is installed on a lower end face part of a second gel formation domain, and first gel solution is filled from a first gel filling opening part (2a) on the side end face and polymerized, to thereby form the gel interface horizontally with excellent reproducibility. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a method for producing an electrophoresis gel cassette, and more particularly to a method for forming a boundary surface between a first gel and a second gel constituting an electrophoresis gel.

  Electrophoresis technology that separates biopolymers such as DNA, RNA, and proteins extracted from animal and plant tissues and cells into electrophoresis gels based on differences in size, properties, etc. is an extremely important technology in the life science field. It is. In particular, when separating proteins by electrophoresis, two-dimensional electrophoresis combining SDS-polyacrylamide gel electrophoresis based on differences in protein molecular weight and isoelectric focusing based on differences in isoelectric points is widely used. It has been.

  In the electrophoresis method as described above, in order to acquire an electrophoretic image with high reproducibility, preparation of a substance to be separated (sample) to be subjected to electrophoresis gel, voltage applied in electrophoresis, current value, time, etc. It is important to set the electrophoresis conditions and to create a stable and highly reproducible gel for electrophoresis.

JP 2007-64848 A

  In a conventional method, when preparing an electrophoresis gel, first, a first gel (separation gel) for separating and developing a protein sample is poured into an electrophoresis gel cassette and filled, and the upper end surface is filled with an isolator. A gel end face is formed horizontally by overlaying butanol or water. Following separation gel polymerization, a gel for electrophoresis is prepared by filling and polymerizing a second gel (concentrated gel) for concentrating the protein sample. However, in order to form the separation gel interface horizontally, an operation of overlaying isobutanol or water must be carefully performed. If the layers are accidentally layered, the interface will be disturbed and will not be horizontal, and the interface will be unevenly polymerized.

  In addition, when water is used as a solvent for layering, there is a high possibility of mixing with the filled separation gel solution. Therefore, skill is required to form the gel interface horizontally with good reproducibility, and the operation is troublesome. In addition, it is necessary to remove the loaded isobutanol or water after the separation gel polymerization, but since the droplets of the solution remain on the wall surface of the filling container, it must be washed with water and absorbed with a paper towel or the like to remove it. Don't be.

  In addition, as a method of filling the electrophoresis gel, as described above, the separation gel is first filled in the electrophoresis gel cassette, the interface is arranged and polymerized, and then the concentrated gel solution is filled and polymerized. Apart from this, it is also possible to carry out a continuous filling method in which the concentrated gel solution is continuously filled and polymerized immediately after filling the separated gel solution. In this continuous filling method, it is possible to save the trouble of overlaying isobutanol or water, but it is necessary to carefully overlay the concentrated gel solution on the previously filled separation gel so as not to disturb the interface. Since skill is required, it is difficult to produce a gel with good reproducibility.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a method for producing a gel cassette for electrophoresis that can form a packed separation gel interface horizontally and accurately. is there.

The present invention relates to a method for producing a gel cassette for electrophoresis, and the invention according to claim 1 is a substrate, another substrate facing the substrate, the substrate and the other substrate. A gel forming region for electrophoresis, and a method for producing an electrophoresis gel cassette comprising a two-layer electrophoresis gel comprising a first gel and a second gel formed in the gel forming region. A step of installing a shielding structure in the gel formation region, a step of filling the gel formation region with the first gel, a step of removing the shielding structure, and the gel formation region for electrophoresis. A method of manufacturing a gel cassette for electrophoresis , wherein the shielding structure is a balloon-like shielding structure .

The invention according to claim 2, in the step of placing the balloon-like shielding structure to the gel-forming region, placing the balloon-like shielding structure in a region filled with the second gel The method for producing a gel cassette for electrophoresis according to claim 1.

The invention according to claim 3, wherein the gel-forming region in the process of installing the balloon-like shielding structure, inserts placed in the upper end surface of the electrophoresis gel cassettes to the balloon-shaped shield structure It is a manufacturing method of the gel cassette for electrophoresis of Claim 1 or 2 characterized by the above-mentioned.

The invention according to claim 4 is the step of installing the balloon-shaped shielding structure in the gel forming region, wherein the first gel and the second gel on the surface of the gel cassette substrate for electrophoresis The gel cassette for electrophoresis according to claim 1 or 2, wherein the balloon-like shielding structure is inserted and installed in a direction perpendicular to the surface of the gel cassette substrate from a portion located at the boundary portion of the gel cassette. It is a manufacturing method.

  Prepare an electrophoresis gel cassette with an opening for filling the gel solution for electrophoresis, install a shielding structure at the position where the interface between the separation gel solution and the concentrated gel solution is formed, and remove the separation gel. Filling from the opening for gel filling, sealing the opening, and allowing to stand allows the separated gel to be polymerized to form the boundary surface horizontally.

  In a gel cassette for electrophoresis, by installing a shielding structure at the interface between the separation gel solution and the concentrated gel solution, a solvent such as isopropanol or water is applied to the upper end surface of the filled separation gel solution as in the existing technology. It is possible to form the electrophoresis gel boundary surface horizontally and with good reproducibility by simply filling the separation gel solution from the gel filling opening without overlaying, sealing the opening, and allowing to stand. Become. Moreover, it is only an operation of installing the shielding structure in the gel cassette and removing it after forming the separated gel, and it is easy to perform without using a skilled operation such as carefully overlaying a solvent for forming an interface by a known method. Can be done.

It is a figure which shows the formation method of the gel cassette for electrophoresis of an example of this invention. It is a figure which shows the formation method of the gel cassette for electrophoresis of an example of this invention. It is a figure which shows one Embodiment of the manufacturing method of the gel cassette for electrophoresis of this invention. It is a figure which shows one Embodiment of the manufacturing method of the gel cassette for electrophoresis of this invention. It is a figure which shows one Embodiment of the manufacturing method of the gel cassette for electrophoresis of this invention. It is a figure which shows one Embodiment of the manufacturing method of the gel cassette for electrophoresis of this invention.

Hereafter, the manufacturing method of the gel cassette for electrophoresis of this invention is demonstrated. First, a method for forming an electrophoresis gel cassette according to an example of the present invention will be described. 1 to 3, FIG. 5, and FIG. 6 are reference examples.

  An example of the structure of the gel cassette for electrophoresis used in the present invention is shown in FIG. The electrophoresis gel cassette comprises two electrophoresis gel cassette substrates (1a) and an electrophoresis gel cassette substrate (1b) facing each other, and the electrophoresis gel cassette substrate (1a) and electrophoresis gel cassette The gel cassette spacer (1c) for electrophoresis is sandwiched so as to be positioned at the end between the gel cassette substrate (1b). By sandwiching the electrophoresis gel cassette spacer (1c), a gel forming region is secured between the electrophoresis gel cassette substrate (1a) and the electrophoresis gel cassette substrate (1b). At the time of filling the gel solution, an end face sealing tape (3) is attached to the lower end surface of the electrophoresis gel cassette, and the shielding structure (4) is filled with the second gel in the gel forming region of the electrophoresis gel cassette. Install at the lower end of the area. After filling the first gel, the first gel filling opening sealing tape (5a) is applied to the first gel filling opening (2a) on the side end face of the electrophoresis gel cassette substrate 1 To seal. The second gel filling opening sealing tape (5b) attached to the second gel filling opening (2b) on the side end face of the electrophoresis gel cassette substrate 1 is the first gel polymerization. The shield structure (4) is removed later, and the second gel is filled and then used for sealing and sealing.

  Moreover, you may use the gel cassette for electrophoresis of another form as shown in FIG. As shown in FIG. 2, the flat shielding structure (4c) is installed in the shielding structure insertion port (8) on the surface of the gel cassette substrate for electrophoresis. The shielding structure insertion port (8) has a structure in which a flat shielding structure (4c) can be inserted in a direction perpendicular to the electrophoresis gel cassette substrate surface. After filling the first gel, the flat shield structure (4c) is removed, and the shield structure insertion portion sealing tape (9) is attached and sealed. As shown in FIG. 2, the shielding structure insertion port (8) may be provided on the electrophoresis gel cassette substrate (1a) or on the electrophoresis gel cassette substrate (1b). Further, since the shielding structure insertion port (8) is an insertion port for inserting the flat shielding structure (4c) in order to accurately and horizontally fill the first gel, the gel cassette for electrophoresis It is preferable to provide in the part located in the boundary part of the 1st gel and 2nd gel of a base-material surface.

(Gel cassette for electrophoresis)
As the material constituting the gel cassette base material for electrophoresis (1a) and (1b) according to the present invention, materials such as known glass, acrylic such as PMMA, high-density polyethylene, polyethylene terephthalate, polypropylene, and polystyrene can be used. However, the material is not limited to these as long as the material does not affect gel formation and electrophoresis.

  Further, the gel filling opening (2) installed in the electrophoresis gel cassette of the present invention is installed on one side end surface of the electrophoresis gel cassette as shown in FIGS. 1 and 2, You may install in the other side end surface. In addition, the first gel filling opening (2a) and the second gel filling opening (2b) are not necessarily installed on the same one side end surface. For example, the first gel filling opening ( 2a) may be provided on the left side end face, and the second gel filling opening (2b) may be provided on the right side end face. Or the opening part (2) for gel filling may be installed in the edge part of the gel cassette base material (1a) and (1b) for electrophoresis, and it is for some gel fillings like FIG. 5 mentioned later. The opening may be omitted, and the upper end surface of the electrophoresis gel cassette may be used as the gel filling opening.

  As is well known, the electrophoresis gel cassette substrate (1a) and (1b) and the electrophoresis gel cassette spacer (1c) constitute not only an electrophoresis gel cassette, but also Patent Document 1. It is possible to use an electrophoresis buffer tank integrated type as shown in FIG.

(Shielding structure)
As long as the shielding structure according to the present invention is a material that does not hinder gel formation, a plate made of a material such as plastic, silicon, urethane, or rubber can be used. In particular, a highly flexible material such as silicon, urethane, or rubber is suitable because it can be installed and removed not only from the upper end surface but also from the opening for gel filling.

  In addition, since the shielding structure is used for filling the first gel horizontally and accurately, the shape of the shielding structure can be set in the gel forming region and is a portion in contact with the first gel. Should be horizontal. Specifically, an L-shaped plate-shaped shielding structure (4a) as shown in FIG. 3 to be described later, a plate-shaped shielding structure (4c) as shown in FIG. In the case of using a balloon-like structure as shown in FIG. 4 to be described later, it is only necessary that the portion in contact with the first gel in the expanded state is horizontal.

  In particular, by using a balloon-like structure, the expanded state can be maintained when the gel solution is filled, and the installation or removal can be performed smoothly by bringing the gel solution into a contracted state. Therefore, it is more preferable.

(Sealing tape)
As the material of the sealing tape (3), (5), (9) according to the present invention, any of rubber, acrylic, silicon and urethane can be used. The adhesive is not particularly limited as long as it has high adhesion and sealing properties, but from the viewpoint of preservability of the filled gel cassette for electrophoresis and the storage stability of the filled gel cassette for electrophoresis, an aqueous solvent is used. It is more preferable to use a material that is resistant to heat and can be easily attached and detached immediately before use.

(Electrophoresis gel)
The first gel and the second gel, which are electrophoresis gels according to the present invention, are separated on the basis of differences in size, properties, etc. of molecules contained in the sample by electrophoresis in the gel. And may be referred to simply as a gel. In the gel for electrophoresis, polymer molecules form a complicated network structure so that the sample can move in the gel. Conventionally generally used gel materials such as polyacrylamide or agarose can be suitably used as the main material constituting the gel.

  Here, the first gel of the present invention specifically refers to a separation gel for separating and developing a protein sample, and the second gel of the present invention specifically refers to a protein sample. This refers to a concentrated gel for concentration. In the present invention, an embodiment is shown in which the first gel is filled first, and then the second gel is filled. This is based on the premise that the first gel has a higher specific gravity than the second gel. Yes. On the other hand, when the first gel having a higher specific gravity than the second gel is filled after the second gel is filled, they are mixed with each other, and the boundary surface between the first gel and the second gel is horizontal. Cannot be formed. The first gel and the second gel of the present invention need only have a specific gravity higher than that of the second gel. For example, glycerol or sucrose may be added to increase the specific gravity to satisfy this condition. The first gel may be a concentrated gel and the second gel may be a separated gel.

  Next, an embodiment of the method for producing an electrophoresis gel cassette of the present invention will be described.

  FIG. 3 shows an embodiment of a method for producing an electrophoresis gel cassette using the electrophoresis gel cassette of the present invention. First, the lower end surface of the electrophoresis gel cassette (1) having the gel filling opening (2) on the side surface is sealed with an end surface sealing tape (3) (FIG. 3a). Next, an L-shaped plate-shaped shielding structure (4a) is inserted from the upper end surface of the gel cassette for electrophoresis (1), and placed on the boundary surface between the first gel and the second gel (FIG. 3b). )). Then, the prepared first gel solution (6) is filled in the direction of the arrow (FIG. 3c)), and the first filling opening (2a) is sealed with the first gel filling opening sealing tape (5a). Is sealed and allowed to stand to polymerize the first gel (FIG. 3d). By installing the L-shaped plate-shaped shielding structure (4) in this way, the first gel interface can be formed uniformly and linearly.

  After the first gel is polymerized, the L-shaped plate-shaped shielding structure (4a) is removed (FIG. 3e)), and the upper end surface of the electrophoresis gel cassette is sealed with the end surface sealing tape (3). The second gel solution (7) prepared in the direction of the arrow in the same manner as described above is filled from the second gel filling opening (2b) at the upper part of the side end surface (FIG. 3f)) (FIG. 3f). 3g)), sealing the second gel filling opening (2b) with the second gel filling opening sealing tape (5b) and allowing to stand to polymerize the second gel (FIG. 3h) ).

  In the above example, the L-shaped plate-shaped shielding structure (4a) is used as the shielding structure, but the balloon-shaped shielding structure (4b) is used as shown in FIG. It is also possible to fill the gel solution for electrophoresis in the same manner as in (Fig. 4a) to h)). When using a balloon-like shielding structure (4b), after creating a gel cassette for electrophoresis, it is placed in a region where the second gel is filled in a contracted state, and an inert gas such as nitrogen is introduced into the balloon. It can be sealed by filling and expanding. After the polymerization of the first gel filled in the gel cassette for electrophoresis, it is more preferable because the inert gas filled in the balloon is pulled out and contracted so that it can be easily attached and detached.

  Alternatively, as shown in FIGS. 5 a) to 5 g, the electrophoresis gel cassette may have a form in which the second gel filling opening (2 b) is omitted. In this case, the separation gel is filled and polymerized in the same manner as in FIG. 3, and then the L-shaped plate-shaped shielding structure (4a) is removed (FIG. 5e). 2 (FIG. 5f)). Then, the upper end surface is sealed with an end surface sealing tape (3) and allowed to stand to polymerize the second gel (7) (FIG. 5g)). When such an electrophoresis gel cassette without the second gel filling opening (2b) is used, the second gel filling opening sealing tape (5b) is not necessary, and the labor of the process can be saved. As in FIG. 4, a balloon-like shielding structure (4b) may be used instead of the L-shaped plate-like shielding structure (4a).

  FIG. 6 shows an embodiment of a method for manufacturing an electrophoresis gel cassette using the electrophoresis gel cassette shown in FIG. A flat shielding structure (4c) is previously installed in the shielding structure insertion port (8) on the surface of the electrophoresis gel cassette substrate (FIG. 6b)), and the first method is the same as in FIGS. The gel is filled and polymerized. After the polymerization of the first gel, the flat shield structure (4c) is removed (FIG. 6e), and the shield structure insertion portion sealing tape (9) is applied and sealed (FIG. 6f)). In the same manner, the second gel is filled and polymerized (FIG. 6g) and h)).

  Regarding the gel filling method in the method for producing an electrophoresis gel cassette according to the present invention, a shielding structure is provided at the gel forming region of the prepared electrophoresis gel cassette or at the boundary between the first gel and the second gel. An object may be installed and the gel filling opening of the gel cassette for electrophoresis may be placed upright so as to stand upright and be filled without introducing bubbles, or may be filled obliquely. Immediately after filling the first gel, the gel filling opening is sealed with a sealing tape, so that the gel cassette for electrophoresis is set up vertically as it is or left to stand horizontally to polymerize the gel. Also good.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.

  If the present invention is used, the interface of the electrophoresis gel can be easily formed horizontally and can be prepared with good reproducibility. Therefore, it is possible to obtain an electrophoretic image with good reproduction. Furthermore, by further developing research on biopolymers such as DNA, RNA, and proteins, it can contribute to the development of industries in the fields of medicine, biology, and chemistry.

1. Gel electrophoresis cassette 1a Electrophoresis gel cassette base 1b Electrophoresis gel cassette base 1c Electrophoresis gel cassette spacer 2 Gel filling opening 2a First gel filling opening 2b, second gel filling opening 3, end face sealing tape 4, shielding structure 4a, L-shaped plate-like shielding structure 4b, balloon-like shielding structure 4c, flat-plate-like shielding structure 5 -Gel filling opening sealing tape 5a-First gel filling opening sealing tape 5b-Second gel filling opening sealing tape 6-First gel 7-Second gel 8-Shielding Structure insertion slot 9 / shielding structure insertion part sealing tape

Claims (4)

A substrate;
Another substrate facing the substrate;
A gel-forming region between the substrate and the other substrate;
A method for producing an electrophoresis gel cassette comprising a two-layer electrophoresis gel comprising a first gel and a second gel formed in the gel-forming region,
Installing a shielding structure in the gel-forming region;
Filling the gel-forming region with the first gel;
Removing the shielding structure;
Filling the second gel in the gel formation region for electrophoresis;
And the shielding structure is a balloon-like shielding structure . In the step of placing the balloon-like shielding structure to the gel-forming region, electrical of claim 1, wherein placing the balloon-like shielding structure in a region filled with the second gel A method for producing a gel cassette for electrophoresis. In the step of placing the balloon-like shielding structure to the gel-forming region, to claim 1 or 2, characterized in that the balloon-like shielding structure was inserted in the upper end surface of the electrophoresis gel cassette loading The manufacturing method of the gel cassette for electrophoresis as described. In the step of placing the balloon-like shielding structure to the gel-forming region, a portion located at the boundary between the first gel for electrophoresis gel cassettes substrate surface and the second gel, the balloon The method for producing a gel cassette for electrophoresis according to claim 1 or 2, wherein the shielding structure in a shape is inserted and installed in a direction perpendicular to the surface of the gel cassette substrate.