JP2011039037A - Electrophoresis gel cassette and method for manufacturing precast gel cassette - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing an electrophoresis gel cassette and a precast gel cassette, capable of forming a gel interface horizontally with good repeatability. <P>SOLUTION: The method for manufacturing includes the steps of assembling a first electrophoresis gel cassette base material (1a) and a second electrophoresis gel cassette base material (1b) with an electrophoresis gel cassette spacer (1c) placed between the two base materials, installing a groove structure (2) on the boundary surface between a first gel and a second gel on the formation surface of the electrophoresis gel cassettes sealed by the gel cassette spacer with an end surface sealing tape (3) stuck to the low end surface, and filling and polymerizing a first gel solution to the height to form the gel interface uniformly and linearly. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to an electrophoresis gel cassette and a method for producing a precast gel cassette using the electrophoresis gel cassette, and more specifically, formation of a boundary surface between a first gel and a second gel constituting the electrophoresis gel. It is about the method.

  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 obtain 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 electrophoresis conditions and to create a stable and highly reproducible gel for electrophoresis.
Conventionally, in order to form a gel for electrophoresis, the experimenter himself prepared a gel solution of the desired composition and filled the gel solution into the container-shaped electrophoresis gel cassette formed of resin, glass, etc. Then, the gel was polymerized.
In recent years, it has been required to analyze many samples accurately in a short time. For this reason, it is known to use a precast gel cassette preliminarily molded into a composition and shape necessary for electrophoresis, in order to save the labor of creating a gel by the experimenter and to suppress variation in gel quality. .

JP 2007-64848 A

  In the conventional method, when preparing a gel for electrophoresis, first, a first gel (separation gel) for separating and developing a sample is poured and filled, and then isobutanol or water is overlaid thereon. The gel end face is formed horizontally. 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 mistakenly vigorously 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 problems, and an object of the present invention is to provide a gel cassette for electrophoresis capable of forming a filled separation gel interface horizontally and accurately, and a method for producing a precast gel cassette. Is to provide.

  The electrophoresis gel cassette of the present invention is arranged in such a manner that at least a part of the first electrophoresis gel cassette substrate formed in a plate shape is spaced apart from the first electrophoresis gel cassette substrate. A plate-like second electrophoresis gel cassette base material, and the first electrophoresis gel cassette base material and the second electrophoresis gel cassette base material are different from each other. An electrophoresis gel cassette serving as an electrophoresis gel forming region for filling a gel solution and a second gel solution, wherein the first electrophoresis gel cassette substrate and the second electricity In each of the electrophoresis gel cassettes, at least one of the surfaces defining the electrophoresis gel forming region and facing each other extends in a direction along the surface and perpendicular to the migration direction, and the first gel solution Will be filled A groove structure or a ridge structure that separates the gel forming area for electrophoresis is formed into a gel forming area and a second gel forming area filled with the second gel solution. This is a gel cassette for electrophoresis.

In addition, the electrophoresis gel cassette of the present invention defines the electrophoresis gel forming region and faces each other in each of the first electrophoresis gel cassette substrate and the second electrophoresis gel cassette. It is preferable that the groove structure is formed in each.
Further, the electrophoresis gel cassette of the present invention defines the electrophoresis gel forming region in each of the first electrophoresis gel cassette substrate and the second electrophoresis gel cassette, and faces each other. The protruding line structure may be formed on each of the above.

  Moreover, the manufacturing method of the precast gel cassette of this invention is a manufacturing method of the precast gel cassette using the gel cassette for electrophoresis of this invention, Comprising: Said 1st gel solution is filled with the said 1st gel formation area | region. And a second filling step of filling the second gel-forming region with the second gel solution. A method for producing a precast gel cassette, comprising:

  By installing a groove structure or a ridge structure on the boundary surface between the separation gel for electrophoresis and the concentrated gel of the present invention, filling the separated gel solution to a height with the groove structure or the ridge structure, polymerizing it, isopropanol, water, etc. The gel interface is horizontally formed in the groove structure or the protruding line structure without installing the solvent or the shielding structure.

In the gel cassette for electrophoresis, by installing a groove structure or ridge structure on the boundary surface between the separation gel and the concentrated gel, the electrophoresis gel can be used without overlaying a solvent such as isobutanol or water as in the existing technology. It is possible to form the boundary surface horizontally and with good reproducibility. In addition, there is no need to install a shielding structure at the gel interface, and the gel solution is simply filled into the electrophoresis gel cassette in consideration of preventing air bubbles from entering the gel interface. This process becomes very simple.
Further, according to the method for producing a precast gel cassette of the present invention, the boundary surface between the separation gel and the concentrated gel is formed horizontally with good reproduction by the groove structure or the ridge structure installed on the boundary surface between the separation gel and the concentration gel. can do. For this reason, the defect rate when manufacturing a precast gel cassette can be suppressed.
Furthermore, according to the precast gel cassette manufactured by the manufacturing method, the boundary surface between the separation gel and the concentrated gel can be horizontally formed with a groove structure or a ridge structure, so that this precast gel cassette is used. Thus, the possibility of obtaining a sharp band pattern when electrophoresis is performed can be further increased. As a result, the reliability and detection sensitivity of analysis using electrophoresis can be improved.

It is a figure which shows the structure of the gel cassette for electrophoresis of one Embodiment of this invention. It is a figure which shows the outline | summary of the manufacturing method of the precast gel cassette of one Embodiment of this invention. a) is a front view showing the configuration of a modified example of the electrophoresis gel cassette of the present invention, b) is a cross-sectional view taken along line AA of a), and c) is a precast gel using the electrophoresis gel cassette. It is explanatory drawing for demonstrating the process of manufacturing a cassette. a) to c) are diagrams showing the results of a simulation in which electrophoresis is performed using a precast gel cassette manufactured using the gel cassette for electrophoresis of the present invention, and a) is a groove structure and a ridge structure. The figure which shows the electric force line at the time of simulating in the case of performing electrophoresis by the precast gel cassette which does not have, The electric force line at the time of carrying out the same simulation in the precast gel cassette which has a groove structure The figure shown, c) is a figure which shows the electric line of force when carrying out the same simulation in the precast gel cassette which has a protruding line structure. The figure for demonstrating the Example which manufactures a precast gel cassette using the gel cassette for electrophoresis of the embodiment, and b) provided the groove | channel structure in the precast gel cassette manufactured in the Example. It is the photograph which expanded and showed the part. a) and b) are diagrams for explaining another embodiment of the present invention, and a) is a side sectional view showing the structure of a precast gel cassette manufactured using the gel cassette for electrophoresis of the present invention, b ) Is a photograph showing the results of electrophoresis using the precast gel cassette of the same example. a) is a side sectional view showing the structure of a general precast gel cassette used as a comparative example for the Example, and b) is a photograph showing the result of electrophoresis using the precast gel cassette of the comparative example.

  Hereinafter, a gel cassette for electrophoresis according to an embodiment of the present invention will be described. As shown in FIG. 1, a first electrophoresis gel cassette substrate (1a) having a groove structure (2) on the boundary surface between the first gel and the second gel, and a second electrophoresis gel cassette Prepare the substrate (1b), assemble it with the gel cassette spacer (1c) for electrophoresis so that it is located at the end between them, and secure the gel formation area for electrophoresis by installing the gel cassette for electrophoresis. Let it form. When the gel solution is filled, an end face sealing tape (3) is attached to the lower end surface of the electrophoresis gel cassette.

(Gel cassette for electrophoresis)
The materials of the first and second electrophoresis gel cassette bases (1a, 1b) constituting the electrophoresis gel cassette according to the present invention are known glass, acrylic such as PMMA, high density polyethylene, polyethylene terephthalate, Materials such as polypropylene and polystyrene can be used, but the material is not limited to these as long as it does not affect gel formation and electrophoresis.

  Further, as is well known, not only is the electrophoresis gel cassette constituted by the first and second electrophoresis gel cassette base materials (1a, 1b) and the electrophoresis gel cassette spacer (1c). An electrophoretic buffer tank integrated type as disclosed in Patent Document 1 may be used.

(Groove structure)
The groove structure (2) according to the present invention is located at the boundary between the first gel and the second gel filled in the electrophoresis gel forming region, and is straight on the inner surface of the electrophoresis gel cassette substrate. It is formed in a shape. Uniform formation of the interface can be achieved by filling the electrophoresis gel cassette with the first gel solution up to the height of the groove structure (2) and polymerizing it. The groove structure (2) may be formed symmetrically only on one or both of the inner surfaces of the first and second electrophoresis gel cassette substrates (1a, 1b). However, when the material of the first and second electrophoresis gel cassette substrates (1a, 1b) is hydrophobic, the inner surface of the first and second electrophoresis gel cassette substrates (1a, 1b) It is preferable to install only one of them because the hydrophobicity of the other smooth surface acts to influence the gel interface formation. The width and depth of the groove structure (2) are not particularly limited as long as they do not affect electrophoresis. In addition, when an electrophoresis gel cassette substrate made of a hydrophobic material is used, either the inner surface of the electrophoresis gel cassette substrate or the groove structure portion or both of them may be hydrophilized. .

(Sealing tape)
As the material of the end face sealing tape (3) 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. It may be simply called a gel. In the gel for electrophoresis, polymer molecules form a complicated network structure so that the sample can move in the gel. As a main material constituting the gel, a gel material conventionally used for electrophoresis, such as polyacrylamide or agarose, can be suitably used.

  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, the manufacturing method of the gel cassette for electrophoresis of this invention is demonstrated. The manufacturing method of the gel cassette for electrophoresis which concerns on this invention is shown in FIG. After sealing the lower end surface of the electrophoresis gel cassette (1) shown in FIG. 1 with the end surface sealing tape (3) (FIG. 2a)), the prepared first gel (4) is used as the electrophoresis gel cassette. (Fig. 2b)) and filled up to the height of the groove structure (2) formed in the electrophoresis gel cassette (Fig. 2c)). Even if only this operation is performed, the first gel (4) surface is leveled by allowing to stand and polymerizing, and then the gel interface can be uniformly formed even if the second gel is filled. Therefore, it is not necessary to overlay a solvent such as water after filling the first gel solution, or to insert and install a shielding structure in the second gel formation region, which leads to simplification of the process.

  As described above, by using the electrophoresis gel cassette and the method for producing an electrophoresis gel cassette according to the present invention, the first gel solution is filled up to the height of the groove structure installed inside the electrophoresis gel cassette, and polymerization is performed. By doing so, it is possible to form a uniform gel interface. Therefore, it is possible to eliminate complicated processes in the process up to the formation of a series of electrophoresis gels, reduce processing time and labor, and realize stable production of electrophoresis gels with good reproducibility. is there.

(Modification)
Next, a configuration of a modified example of the electrophoresis gel cassette of the present embodiment will be described with reference to FIG. FIG. 3 is a diagram showing the configuration of the electrophoresis gel cassette (10) of the present modification, a) is a front view showing the configuration of the modification of the electrophoresis gel cassette of the present invention, and b) is a). It is sectional drawing in the X1-X1 line | wire. In the following description, elements having the same configuration as that of the electrophoresis gel cassette (1) described in the above embodiment are denoted by the same reference numerals, and redundant description is omitted.

  As shown in FIG. 3a) and FIG. 3b), the gel cassette for electrophoresis (10) is provided with a convex structure (12) instead of the groove structure (2) in the above-described embodiment. The configuration is different from the gel cassette for electrophoresis (1).

The ridge structure (12) is a plate-like electrophoresis gel cassette substrate (1) formed of the same material as the first and second electrophoresis gel cassette substrates (1a, 1b) in the above-described embodiment. 11a and 11b) are linearly formed at the same positions as in the groove structure (2). That is, the ridge structure (12) extends along each of the opposing surfaces of the electrophoresis gel cassette base material (11a, 11b) and is indicated by an arrow P in the electrophoresis direction (FIG. 3a) of the electrophoresis gel cassette 10. Extending in a direction orthogonal to the direction shown).
In addition, the ridge structure (12) formed on the electrophoresis gel cassette substrate (11a) is formed so as to protrude toward the electrophoresis gel cassette substrate (11b), and the electrophoresis gel cassette substrate The ridge structure (12) formed in (11b) protrudes toward the gel cassette base material (11a) for electrophoresis. In the present embodiment, the ridge structures (12) are arranged facing each other.

  The ridge structure (12) is preferably installed on both the gel cassette base materials (11a, 11b) for electrophoresis, similarly to the groove structure (2) described above. The gel cassette base material for electrophoresis (11a, 11b) may be formed only on one of the inner surfaces.

  When the precast gel cassette (10A) is manufactured using the electrophoresis gel cassette (10) having the above-described configuration, first, the electrophoresis gel cassette base material is set so that the protruding structures (12) face each other. (11a, 11b) are assembled with the gel cassette spacer (1c) for electrophoresis sandwiched therebetween. Furthermore, the end surface sealing tape (3) is affixed to the lower end surface of the gel cassette for electrophoresis as in the above embodiment. Thus, an electrophoresis gel forming region A for filling the electrophoresis gel is formed between the electrophoresis gel cassette substrate (11a) and the electrophoresis gel cassette substrate (11b). In addition, the gel forming region A for electrophoresis is a first gel for filling the first gel solution on the lower end surface side of the gel cassette base material for electrophoresis (11a, 11b) rather than the ridge structure (12). It becomes the formation region A1, and the upper end surface side of the gel cassette base material for electrophoresis (11a, 11b) becomes the second gel formation region A2 for filling the second gel solution with respect to the ridge structure (12). Thus, in this embodiment, the gel formation region A for electrophoresis is divided into two regions by the convex structure (12).

  Next, the gel gel base material for electrophoresis (11a, 11b) is arranged so that the first gel forming region A1 is on the bottom and the second gel forming region A2 is on the top, so that the gel for electrophoresis is formed. In the region A, the first gel solution is injected into the first gel formation region A1. In the present embodiment, the first gel solution is poured into the first gel formation region A1 through the second gel formation region A2 using, for example, a filling machine. At this time, as shown in FIG. 3c), the surface of the gel is positioned at the upper end of the ridge structure (12), and the surface of the first gel solution is substantially aligned along the upper end side corner of the ridge structure (12). Position horizontally.

Next, the second gel solution is overlaid on the first gel solution. If the specific gravity of the second gel solution is lower than that of the first gel container, the second gel solution can be overlaid on the first gel solution before the first gel solution is polymerized. If the second gel solution has a specific gravity lower than that of the first gel solution, the first gel solution is polymerized and then the second gel solution is layered on the first gel solution. Good.
After the second gel solution is overlaid on the first gel solution, the electrophoresis gel cassette (10) is allowed to stand to polymerize the first gel solution and the second gel solution. When the polymerization of the first gel solution and the second gel solution is completed, the polymerized second gel 5 is laminated on the polymerized first gel 4 as shown in FIG. 3c). This completes the production of the precast gel cassette (10A), and proceeds to a subsequent process such as sealing the precast gel cassette (10A) in an appropriate package that prevents drying.

  The manufacturing method of the electrophoresis gel cassette (10) and the precast gel cassette (10A) having such a configuration can achieve the same effects as described in the above embodiment.

Next, a simulation was performed in which electrophoresis was performed using the precast gel cassettes (1A, 10A) manufactured using the electrophoresis gel cassettes (1, 10) described in the above-described embodiments and modifications. The results will be described.
FIG. 4a) is a diagram showing electric lines of force when a simulation is performed when electrophoresis is performed with a precast gel cassette having no groove structure (2) and ridge structure (12), and FIG. 4b) is a groove. FIG. 4c is a diagram showing electric lines of force when a similar simulation is performed in the precast gel cassette (1A) having the structure (2). FIG. 4c) is a similar simulation in the precast gel cassette (10A) having the ridge structure (12). It is a figure which shows an electric force line when doing.

  In this simulation, in order to investigate the influence of the groove structure (2) formed on the electrophoresis gel cassette (1) or the ridge structure (12) formed on the electrophoresis gel cassette (10) on the electrophoresis. The lines of electric force indicating the movement of the potential between the anode electrode and the cathode electrode were visualized.

  As shown in FIG. 4a), in this simulation, the anode electrode 100 and the anode buffer layer 101 are provided on the side where the sample is applied to the electrophoresis gel cassette (the position on the upper end side of the electrophoresis gel cassette in FIG. 1). An attachment portion 102 for attaching a gel strip for two-dimensional electrophoresis is formed, and a cathode electrode 103 and a cathode buffer layer 104 are formed at the end of the electrophoresis gel cassette where the sample is migrated. This is the basic model M0. That is, the gel cassette for electrophoresis that performs this simulation also functions as an electrophoresis tank.

In this simulation, the anode electrode and the cathode electrode are platinum electrodes, and the anode electrode and the cathode electrode can be connected to a power supply device (not shown). In this embodiment, the power supply device is set to apply a DC voltage of 200 V between the anode electrode and the cathode electrode.
In addition, lysozyme protein (molecular weight 14307 Da, isoelectric point 11.1) was adopted as a sample to be subjected to electrophoresis in this simulation. In this simulation, under the above settings, the lines of electric force when lysozyme protein was electrophoresed were set to be displayed on the screen.

  Furthermore, in this simulation, the electrophoresis gel cassette of the basic model M0 and the groove structure (2) having a depth of 0.2 mm and a width of 0.2 mm in addition to the basic model M0 are formed. Gel cassette for electrophoresis (10) (model M1) shown in the cassette (FIG. 4b), and the above-mentioned basic model M0, and a convex strip structure (12) having a height of 0.2 mm and a width of 0.2 mm is formed (FIG. Each model M10) shown in 4c) was set as a model, and simulation was performed for each of the basic model M0, the model M1, and the model M10.

  As a result of this simulation, as shown in FIGS. 4a) to 4c), the electric lines of force in the model M1 in which the groove structure (2) is formed and the model M10 in which the ridge structure (12) is formed are It was almost equivalent to the electric field lines in the basic model M0 described above. From this result, a ridge structure (12) having a height of at least 0.2 mm and a width of 0.2 mm, or a ridge structure (12) smaller than this ridge structure (12), and a depth of 0.2 mm and a width of 0.2 mm. In the gel cassette for electrophoresis in which the groove structure (2) or the groove structure (2) smaller than the groove structure (2) is formed, the groove structure (2) or the ridge structure (2) affects the electric field lines. It is thought that there is almost no influence on the electrophoresis result such as protein retention.

  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.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

Example 1
[1. Materials and methods〕
(1-1. Creation of groove cassette and gel cassette for electrophoresis)
A linear groove structure having a width of 1 mm was formed in the horizontal direction of the gel forming surface above the gel forming surfaces of the two electrophoresis gel cassette substrates made of acrylic resin described in Patent Document 1 described above. Subsequently, these two electrophoresis gel cassette substrates were prepared by welding with an ultrasonic welding apparatus.

(1-2. Composition of gel for electrophoresis)
A polyacrylamide gel solution containing 13% or 4% acrylamide, 375 mM Tris-HCl / pH 8.8, 0.1% ammonium persulfate, and 0.1% tetramethylethylenediamine was used. Add 29.2% acrylamide-0.8% methylenebisacrylamide mixture so that the final concentration of acrylamide in the polyacrylamide gel solution is 13% for the separation gel and 4% for the concentrated gel. Prepared.

(1-3. Packing of electrophoresis gel)
1-1. After sealing the lower end surface of the gel cassette for electrophoresis prepared as shown in Fig. 2, the separation gel solution is filled from the upper end surface to the groove forming part and allowed to stand to polymerize the gel. I let you.

[2. result〕
A gel formation test was carried out using the gel cassette for electrophoresis according to the present invention.

  According to the preparation process shown in FIG. 2, the gel solution was polymerized by filling the gel gel for electrophoresis up to the groove forming portion and allowing it to stand. The result is shown in FIG. In the schematic diagram shown in FIG. 5a), a photograph enlarging a portion surrounded by a broken line portion, that is, a separation gel interface portion provided with a groove structure is shown in FIG. 5b). Moreover, the part of the both ends of a gel interface is shown with an arrowhead. In the gel cassette provided with the groove forming part, it was shown that the interface of the filled and polymerized separated gel was uniformly formed (FIG. 5b) 1). On the other hand, when using a conventional gel cassette in which no groove forming part was installed on the gel forming surface, the separation gel upper end interface was affected by the hydrophobicity of the gel cassette material, resulting in a severely uneven shape ( FIG. 5b) 2).

(Example 2)
Next, for Example 2 in which the precast gel cassette (1A) is produced using the electrophoresis gel cassette (1) having the groove structure (2), the precast gel cassette (1A) produced in Example 2 is used. Then, it explains with the result of having performed electrophoresis. As a comparative example for the present example, an example of producing a precast gel cassette having no groove structure (2) using a general electrophoresis gel cassette having no groove structure (2) is shown.
6a) and 6b) are diagrams for explaining Example 2 of the present invention, and FIG. 6a) is a precast gel cassette (1A) produced using the gel cassette for electrophoresis (1) of the present invention. FIG. 6B) is a photograph showing the result of electrophoresis using the precast gel cassette (1A). Fig. 7a) is a side sectional view showing the structure of a general precast gel cassette used as a comparative example with respect to Example 2, and Fig. 7b) shows the result of electrophoresis using the precast gel cassette of the comparative example. It is a photograph.

As shown in FIG. 6a), in this example, the groove structure (2) has a depth of 0.1 mm and a width of 0.1 mm, and each of the electrophoresis gel cassette base materials (1a, 1b). The shape extends along the plane and in a direction orthogonal to the migration direction in which electrophoresis is performed. The thickness of the gel cassette spacer for electrophoresis (1c) was 1 mm. Thereby, the thickness of the gel in parts other than the groove structure (2) is 1 mm.
Moreover, the composition of the gel in a present Example is that the 1st gel solution is a 13% polyacrylamide solution, and the 2nd gel solution is a 4% polyacrylamide solution. That is, in Example 2, the first gel solution is a solution that becomes a separation gel, and the second gel solution is a solution that becomes a concentrated gel.
The composition of the first gel solution is shown in Table 1 below.

  The composition of the second gel solution is shown in Table 2 below.

  In the method for producing the precast gel cassette (1A) in this example, the lower end surface of the produced electrophoresis gel cassette (1) is sealed with the sealing tape (3), and the first gel solution is formed into a groove structure ( After the injection to the height of 2), the second gel solution was continuously injected without overlaying water to polymerize the first gel solution and the second gel solution, respectively.

  Further, as shown in FIG. 7a), as a comparative example with respect to Example 2, the groove structure (2) was not formed, and a 1 mm thick gel having the same composition as in Example 2 was formed inside. A precast gel cassette was produced.

  In addition, a benchmark made by Invitrogen, which is a protein molecular weight marker, was employed as a sample to be electrophoresed using the precast gel cassette produced in Example 2 and its comparative example. This molecular weight marker contains seven kinds of proteins of 155 kDa, 98 kDa, 63 kDa, 40 kDa, 32 kDa, 21 kDa, and 11 kDa in order from high molecular weight to low molecular weight. In addition, as lanes for electrophoresis of molecular weight markers with respect to the respective precast gel cassettes manufactured in Example 2 and its comparative example, lanes L1 to L4 shown in FIG. 6b) and lane L5 shown in FIG. 7b) Thru L8 were set, and 1 μl of molecular weight marker was applied per lane.

  Moreover, as a running buffer for performing electrophoresis, a buffer composed of 25 mM tris, 192 mM Grysin, and 0.2% SDS (all at a final concentration) is used on the cathode side, and on the anode side, A buffer consisting of 150 mM Tris was used.

  Electrophoresis is performed in a constant current mode set at 25 mA for 25 minutes. After the electrophoresis is completed, the gel is taken out from the precast gel cassettes of the present example and the comparative example, and the gel is irradiated with excitation light having an excitation wavelength of 520 nm. Fluorescence images of molecular weight markers were taken.

As a result of performing electrophoresis using the precast gel cassette manufactured by this example and its comparative example, as shown in FIG. 6 b), the electrophoresis gel cassette (1) having the groove structure (2) in Example 2 In the precast gel cassette (1A) produced using the above method, the protein migration pattern was horizontal in each lane. On the other hand, as shown in FIG. 7b), when an electrophoresis gel cassette having no groove structure (2) is used, the protein migration pattern appears to wave in each lane. It became uneven.
Moreover, as a result of performing electrophoresis using 4 lanes in the precast gel cassette of this example and the comparative example, in the precast gel cassette (1A) of this example, the bands composed of proteins of the same molecular weight were each in 4 lanes. In the same distance. That is, a sharp band pattern was obtained with good reproducibility by the precast gel cassette (1A) in which the groove structure (2) was formed.

From these results, it was found that according to the gel cassette for electrophoresis (1) described in the above embodiment, the reliability of analysis using electrophoresis and the detection sensitivity can be improved.
Further, in this embodiment, it is not necessary to overlay water before the second gel solution is overlaid on the first gel solution, so that it is necessary to manufacture the precast gel cassette (1A). The working time was shortened by about 30 minutes compared with the case of overlaying water, and the precast gel cassette (1A) could be manufactured more easily than in the past.

  By using the present invention, the interface of the electrophoresis gel can be formed horizontally, and the electrophoresis gel can be easily and reproducibly produced. 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.

DESCRIPTION OF SYMBOLS 1 * Gel cassette for electrophoresis 1a * Gel cassette base material for 1st electrophoresis 1b * Gel cassette base material for 2nd electrophoresis 1c * Gel cassette spacer for electrophoresis 2 * Groove structure 3 * End face sealing tape 4 First gel 5 Second gel 10 Electrophoresis gel cassette 11a First gel cassette base 11b Second gel cassette base 12 Projection structure A Electric Gel formation region for electrophoresis A1 · First gel formation region A2 · Second gel formation region

Claims (4)

A first electrophoresis gel cassette substrate formed in a plate shape, and a plate-shaped second electrophoresis in which at least a part is arranged apart from the first electrophoresis gel cassette substrate The first gel solution and the second gel solution are different from each other between the first electrophoresis gel cassette substrate and the second electrophoresis gel cassette substrate. An electrophoresis gel cassette to be an electrophoresis gel forming region for filling
In each of the first electrophoresis gel cassette substrate and the second electrophoresis gel cassette, at least one of the surfaces that define the electrophoresis gel forming region and face each other is along the surface and Extending in a direction orthogonal to the migration direction, the first gel formation region filled with the first gel solution and the second gel formation region filled with the second gel solution are used for the electrophoresis. A gel cassette for electrophoresis characterized in that a groove structure or a ridge structure for separating gel forming regions is formed.   In each of the first electrophoresis gel cassette substrate and the second electrophoresis gel cassette, the groove structure is formed on each of the surfaces that define the electrophoresis gel forming region and face each other. The gel cassette for electrophoresis according to claim 1.   In each of the first electrophoresis gel cassette substrate and the second electrophoresis gel cassette, the protruding gel structure is formed on each of the surfaces that define the electrophoresis gel forming region and face each other. The gel cassette for electrophoresis according to claim 1, wherein the gel cassette is used for electrophoresis. A method for producing a precast gel cassette using the gel cassette for electrophoresis according to any one of claims 1 to 3,
A first filling step of filling the first gel formation region with the first gel solution;
A second filling step of filling the second gel formation region with the second gel solution;
A method for producing a precast gel cassette, comprising: