JP5246128B2 - Method for producing gel cassette for electrophoresis and filling method for gel for electrophoresis - Google Patents

Description

  The present invention relates to a method for producing an electrophoresis gel cassette in which a gel cassette body is filled with an electrophoresis gel, and is characterized by a method for forming a boundary surface between a first gel and a second gel constituting the electrophoresis gel. Technology.

  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, a two-dimensional electrophoresis method combining SDS-polyacrylamide gel electrophoresis based on differences in protein molecular weight and isoelectric focusing based on differences in isoelectric points. Is widely used (see Patent Document 1).

In the electrophoresis method as described above, in order to obtain an electrophoretic image with high reproducibility, preparation of a protein sample to be subjected to electrophoresis gel, electrophoresis conditions such as voltage, current value and time applied in electrophoresis Setting is important. Similarly, it is important to create a stable and reproducible gel for electrophoresis.
In an ordinary method, when preparing an electrophoresis gel, first, a first gel (separation gel) for separating and developing a protein sample is poured into a gel cassette body for electrophoresis and filled. Next, the gel end surface is horizontally formed by overlaying isobutanol or water on the upper end surface of the filled first gel. Following separation gel polymerization, a gel for electrophoresis is prepared by filling and polymerizing a second gel (concentrated gel) for concentrating the protein sample.

JP 2007-64848 A

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, after separation gel polymerization, an operation for removing the loaded isobutanol or water is required. For this reason, since droplets of the solution remain on the wall surface of the filling container, they must be washed with water and absorbed with a paper towel or the like to be removed.
Here, as a method for filling the electrophoresis gel, as described above, the separation gel is first filled in the electrophoresis gel cassette, the interface is aligned and polymerized, and then the concentrated gel solution is filled and polymerized. In addition to the method, a continuous filling method in which the concentrated gel solution is continuously filled and polymerized immediately after the separation gel solution is filled can also be implemented. In this continuous filling method, the labor of overlaying isobutanol or water can be saved. However, it is necessary to carefully overlay the concentrated gel solution on the previously filled separation gel so as not to disturb the interface, and skill is required, so that it is difficult to prepare a gel with high 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 an electrophoresis gel cassette capable of forming a filled gel interface horizontally and accurately, and an electrophoresis gel. It is to provide a filling method.

In order to solve the above-mentioned problem, the invention described in claim 1 of the present invention is a method for producing an electrophoresis gel cassette in which a gel cassette body containing a gel is filled with an electrophoresis gel,
The gel for electrophoresis includes a first gel and a second gel having a specific gravity lower than that of the first gel,
A first step of filling the gel cassette body with the first gel, and a second step of filling the gel cassette body with the second gel after the first step;
During the first step, or between the first step and the second step, the gel cassette body is horizontally or intermittently given ultrasonic vibration to the gel cassette body, so that the gel interface of the filled first gel is horizontal or It is characterized by including a process of approaching horizontal.

Next, the invention described in claim 2 is the configuration described in claim 1, in which the vibration caused by the ultrasonic waves is the first filled in the surface, side end surface, lower end surface of the gel cassette body and the gel cassette. It is characterized by being applied to at least one place in the gel solution interface.
Next, in the invention described in claim 3, after the gel cassette body containing the gel is filled with the first gel constituting the electrophoresis gel, the electrophoresis gel is constituted and the specific gravity is higher than that of the first gel. Fill the gel cassette body with a low second gel, and horizontally form the interface of the filled first gel by applying ultrasonic vibration to the gel cassette body before filling the second gel. An electrophoretic gel filling method is provided.

  According to the present invention, the interface of the filled first gel is horizontal or nearly horizontal by simply applying ultrasonic vibration to the gel cassette body for electrophoresis filled with the first gel to be a separated gel solution or the like. It becomes. As a result, it is possible to form the electrophoresis gel interface horizontally, reproducibly and easily without performing a complicated operation of overlaying an aqueous solvent on the upper end surface of the filled first gel. .

It is a figure which shows the structure of the gel cassette for electrophoresis which concerns on embodiment based on this invention. It is a figure which shows one Embodiment of the manufacturing method of the gel cassette for electrophoresis based on embodiment based on this invention. It is a figure which shows embodiment of the method of giving a vibration after filling the 1st gel in the manufacturing method of the gel cassette for electrophoresis based on embodiment based on this invention. It is a figure which shows one Example of the manufacturing method of the gel cassette for electrophoresis based on embodiment based on this invention.

Next, embodiments according to the present invention will be described with reference to the drawings.
(Gel cassette body for electrophoresis)
As shown in FIG. 1, the electrophoresis gel cassette main body (1) of the present embodiment comprises a first electrophoresis gel cassette substrate (1a), a second electrophoresis gel cassette substrate (1b), And a gel cassette spacer (1c) for electrophoresis. First, as shown in FIG. 1, the first electrophoresis gel cassette substrate (1a) and the second electrophoresis gel cassette substrate (1b) are arranged opposite to each other and located at the end between them. By assembling the electrophoresis gel cassette spacer (1c) as described above, the electrophoresis gel cassette main body (1) having the electrophoresis gel forming region secured is obtained.

Further, before the gel solution is filled, the end surface sealing tape (2) is attached and closed to the lower end surface of the electrophoresis gel cassette body (1).
As the material of the first and second gel cassette base materials for electrophoresis (1a, 1b), known materials such as 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.

  The electrophoresis gel cassette main body (1) includes the first and second electrophoresis gel cassette substrates (1a, 1b) each having a plate shape as described above, and an electrophoresis gel cassette spacer ( The electrophoretic gel cassette body (1) is not limited to 1c). As the gel cassette body (1), an electrophoretic buffer tank integrated type as disclosed in Patent Document 1 may be used.

  Furthermore, the material of the gel cassette body for electrophoresis (1) described above may be a hydrophobic material such as an acrylic material, but may be subjected to a hydrophilic treatment. It is more preferable to use the base material of the electrophoresis gel cassette body (1) subjected to the hydrophilization treatment because the disturbance of the end face when the first gel solution is filled can be suppressed.

(Sealing tape)
As the material of the end face sealing tape (2), 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 electrophoresis gel of this embodiment is a gel that is separated based on differences in sample size, properties, and the like by electrophoresis in the gel. This gel for electrophoresis is sometimes simply referred to as 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, a first gel and a second gel are prepared as electrophoresis gels to be filled in the gel cassette body (1).
In the present embodiment, the first gel will be described as an example of a separation gel for separating and developing a protein sample. In addition, the second gel will be described using a concentration gel for concentrating a protein sample as an example. However, the first gel and the second gel of the present embodiment only need to have a specific gravity higher than that of the second gel. Therefore, for example, the first gel may be a concentrated gel and the second gel may be a separated gel if this condition is satisfied by adding glycerol or sucrose to increase the specific gravity.

Here, in the present embodiment, as described later, the gel cassette body (1) is first filled with the first gel and then filled with the second gel. This assumes that the first gel has a higher specific gravity than the second gel. When the first gel having a higher specific gravity than that of the second gel is filled after the second gel is filled first, the boundary between the first gel and the second gel is mixed. The surface cannot be formed horizontally. For this reason, the specific gravity of the second gel is required to be lower than that of the first gel.

(Method for producing gel cassette for electrophoresis)
The gel solution filling method in the electrophoresis gel cassette manufacturing method is performed as follows. The gel filling may be performed according to a known method.
First, as the first step, the prepared first gel solution is filled from the upper end surface of the prepared gel cassette body for electrophoresis (1).

  Next, as an intermediate step, vibration by ultrasonic waves or the like is given continuously or at regular intervals to the gel cassette body (1) filled with the first gel solution. As a result, the gel interface portion is formed horizontally without overlaying an aqueous solvent on the filled first gel solution. The ultrasonic vibration may be performed during the first gel solution filling as long as the filling operation is performed carefully.

As an apparatus for generating the ultrasonic vibration, for example, an ultrasonic cleaner may be used. The apparatus is not limited as long as the apparatus emits an output with an ultrasonic frequency of 40 to 50 kHz. Alternatively, the gel cassette body (1) filled with the separated gel solution may be vibrated by a vortex mixer or the like. Vibration due to these may be continuously applied to the gel cassette immediately after gel filling, or a short interval that does not vibrate may be provided at regular intervals. Alternatively, vibration may be applied while filling the first gel.
After the first gel filled in this way is polymerized, as a second step, the prepared second gel solution is filled from the upper end surface of the electrophoresis gel cassette body (1) according to a known method.

(One embodiment)
Furthermore, an embodiment of a method for producing a gel cassette for electrophoresis will be described.
FIG. 2 is a diagram showing an embodiment of an electrophoresis gel cassette manufacturing method according to the present invention.
First, the lower end surface of the gel cassette body (1) prepared as shown in FIG. 1 is sealed with an end surface sealing tape (2).

  Next, the separated gel solution to be the first gel 3 is filled from the upper end surface of the gel cassette body (1) (FIG. 2a)). In particular, when a hydrophobic material is used as the material of the gel cassette body (1), the gel interface is not generally horizontal, but becomes uneven as shown in FIG. 2b).

Next, the gel cassette main body (1) filled with the separated gel solution to be the first gel 3 is not tilted by vibration force to an apparatus (not shown) that generates ultrasonic waves or vibrations such as an ultrasonic cleaner. Then, by continuously applying vibration (4) (FIG. 2c)), the upper end surface (5) of the filled first gel 3 is uniformly and horizontally polymerized (FIG. 2d)).
Here, FIG. 3 shows an example of a method for applying vibration to the electrophoresis gel cassette filled with the first gel 3.

FIG. 3A) shows a method of applying vibration to the side end face of the electrophoresis gel cassette, and FIG. 3B) is a method of applying vibration to the lower end face of the electrophoresis gel cassette. FIG. 3c) shows a method of applying vibration to the surface of the electrophoresis gel cassette, that is, the surfaces of the first and second electrophoresis gel cassette bases (1a, 1b) constituting the electrophoresis gel cassette. . It is also possible to apply vibration by combining any of these methods, and further combining the methods shown in FIGS. In addition, as shown in FIG. 3d), vibration may be directly applied only to the interface portion of the first gel 3 filled in the electrophoresis gel cassette body (1).
After the first gel is polymerized, the gel cassette body (1) is filled with the second gel solution.

(Effect of this embodiment)
As described above, by using the electrophoresis gel cassette manufacturing method according to the present invention, the electrophoresis gel cassette body (1) is filled with the separation gel solution that is the first gel 3 up to a predetermined height, and is subjected to ultrasonic waves. It is possible to form a horizontal gel interface with respect to the first gel 3 by performing only the operation of giving vibration such as. Therefore, it is possible to eliminate the complicated processing in the process of manufacturing a series of known gel cassettes for electrophoresis, reduce processing time and labor, and realize stable production of electrophoresis gel with good reproducibility. Is possible.

In addition, by applying the vibration by the ultrasonic wave to at least one place in the surface of the gel cassette body, the side end face, the lower end face and the first gel solution interface filled in the gel cassette, as described above, The interface of the first gel 3 can be made horizontal or horizontal.
Here, the present embodiment is not limited to the above-described embodiment, 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.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[1. Materials and methods〕
(1-1. Preparation of gel cassette body for electrophoresis)
It was prepared by welding the lid and substrate member of the gel cassette body described in Patent Document 1 (JP 2007-64848 automated two-dimensional electrophoresis apparatus and apparatus component) with an ultrasonic welding apparatus.

(1-2. Composition of gel for electrophoresis)
A polyacrylamide gel solution containing 10% acrylamide, 375 mM Tris-HCl / pH 8.8, 0.1% ammonium persulfate, and 0.1% tetramethylethylenediamine was used as the first gel solution. Using a 29.2% acrylamide / 0.8% methylenebisacrylamide mixed solution, the final concentration of acrylamide in the polyacrylamide gel was adjusted to 10%.

(1-3. Packing of gel for electrophoresis)
1-1. After sealing the lower end portion of the electrophoresis gel cassette body prepared as shown in FIG. The polyacrylamide gel solution (first gel solution) was filled to a predetermined height.

(1-4. Ultrasonic vibration)
1-3. The gel cassette body filled with the gel solution is fixed to an ultrasonic cleaning device (manufactured by ASONE), ultrasonic waves are generated, and vibration is applied to the lower end surface of the gel cassette body filled with the gel solution and the surface of the gel cassette for 20 minutes. Continued.

(2. Results)
A gel formation test was carried out using the gel cassette for electrophoresis according to the present invention.
In accordance with the preparation process shown in FIG. 2, the electrophoresis gel cassette body (1) is filled to a predetermined height with the electrophoresis separation gel solution, and the first gel is continuously applied with ultrasonic vibration as shown in FIG. (3) was polymerized. The result is shown in FIG.

In the schematic diagram shown in FIG. 4a), the state of the part surrounded by the broken line, that is, the enlarged part of the separated gel interface part is shown in FIG. 4b). In the gel cassette body (1) that continued to be subjected to ultrasonic vibration, it was shown that the interface between the separated and filled separated gel was uniformly formed (see FIG. 4b) -1. On the other hand, in the gel cassette body (1) which is allowed to stand without applying ultrasonic vibration after filling the gel solution, the separation gel upper end interface may be affected by the hydrophobicity of the gel cassette material, but the unevenness is severe. It became a shape (see FIG. 4b) -2).

  Thus, according to the present invention, the interface of the separated gel can be uniformly formed without the hydrophilic treatment of the gel cassette body (1).

  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 Electrophoresis gel cassette body 1a First electrophoresis gel cassette substrate 1b Second electrophoresis gel cassette substrate 1c Spacer 2 End face sealing tape 3 First gel 4 Ultrasonic vibration 5 First The top surface of the gel

Claims (3)

A gel cassette manufacturing method for electrophoresis in which a gel cassette body containing a gel is filled with an electrophoresis gel and manufactured.
The gel for electrophoresis includes a first gel and a second gel having a specific gravity lower than that of the first gel,
A first step of filling the gel cassette body with the first gel, and a second step of filling the gel cassette body with the second gel after the first step,
During the first step, or between the first step and the second step, the gel cassette body is horizontally or intermittently given ultrasonic vibration to the gel cassette body, so that the gel interface of the filled first gel is horizontal or The manufacturing method of the gel cassette for electrophoresis characterized by including the process close | similar to horizontal.   2. The electricity according to claim 1, wherein the vibration due to the ultrasonic waves is applied to at least one place on the surface, side end surface, and lower end surface of the gel cassette body and the first gel solution interface filled in the gel cassette. Method for producing gel cassette for electrophoresis.   After the gel cassette body containing the gel is filled with the first gel constituting the electrophoresis gel, the gel cassette body is filled with the second gel that constitutes the electrophoresis gel and has a specific gravity lower than that of the first gel. Before filling the second gel, the gel cassette body is filled with an ultrasonic vibration to form an interface of the filled first gel horizontally. Method.

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