JPH11211695A - Gel for electrophoresis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent a gel from being broken down in a staining operation by a method wherein a sheetlike rigid body is used as a container material on the side coming into contact with an electrode solution and a water-insoluble material is used as a gel contact face on the other side. SOLUTION: A rectangular gel bonded film is brought into close contact with a glass plate so as to be supported. The film on which a spacer in a thickness of 1 mm is pasted is pressed to a glass plate having a U-shaped cutout on the upper part, and a monomer aqueous solution is injected into a mold which is sealed with silicone rubber so as to prevent the solution from being leaked from the lower part. Then, a comb made of PE is pressed from the U-shaped cutout, it is left for three hours at 27 deg.C, a polymerizing operation is completed, and a gel for electrophoresis is created. The gel is set on an electrophoretic apparatus, and the electrophoretic operation of a sample is performed. After it is finished, the glass plate is removed, and an aqueous gel in a state that it is bonded to the gel bonded film is immersed in a staining solution for one hour and in a bleaching solution all night. In this case, the aqueous gel is not broken down, and an electrophoretic image is clear so as to be suitable for being dried and preserved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gel for electrophoresis used in the field of biochemistry and medicine for the separation and analysis of biological substances, for example, proteins and nucleic acids. The present invention relates to a container or a gel support containing an aqueous gel.

[0002]

2. Description of the Related Art An aqueous gel of crosslinked polyacrylamide or an aqueous gel of crosslinked polyN-vinylcarboxylic acid amide has been awarded as an electrophoresis gel. The electrophoresis speed of proteins and nucleic acids (DNA, RNA, etc.) in gels during electrophoresis is such that the lower the concentration of the aqueous polymer constituting the gel, the larger the pore size, and can cope with the analysis of high molecular weight proteins and nucleic acids. In particular, in the analysis of nucleic acids, it is desired to use an aqueous gel having a low polymer concentration (4.5% or less). In order to prevent the temperature from rising due to the heat generated by the gel during energization, it is general to set the gel vertically, set one side in contact with the electrode solution, and cool the gel with the contacted solution. The gel used in this manner supports the gel in a container such as glass or plastic, and at the same time, performs electrical insulation. A concave cut is made in the upper part of the container wall on the electrode (negative electrode) liquid side, and the aqueous gel and the electrode ( In general, the liquid has a structure in which a liquid is brought into contact with the liquid and a current is supplied in the vertical direction, and a well is provided above the gel for setting an analysis sample. The gel after the electrophoresis is removed from the container, and is subjected to staining as a soft aqueous gel without a support.

[0003]

An aqueous gel having a low polymer concentration is fragile, and has a drawback that the gel is liable to break when a dyeing operation is performed without a soft, bare support. .

[0004]

Means for Solving the Problems The present inventor uses a plate-shaped rigid body having a concave notch on the upper side as a container material on the side to be cooled in contact with the electrode solution, thereby facilitating peeling, and contacting the other gel. The use of a water-insoluble material, such as a gel-bonded film, having an ability to adhere to an aqueous gel for electrophoresis, prevented gel destruction during dyeing and successfully solved the above problem.

According to the first aspect of the present invention, one surface of the planar aqueous gel for electrophoresis having a well is in contact with a water-insoluble material exhibiting an adhering ability (gel bond property) to the aqueous gel for electrophoresis. A gel for electrophoresis, characterized in that:

According to a second aspect of the present invention, the aqueous gel for electrophoresis is produced by radical polymerization of an aqueous monomer solution containing acrylamide and a water-soluble divinyl compound. This is an electrophoresis gel.

The invention of claim 3 of the present invention is characterized in that the aqueous gel for electrophoresis is produced by radical polymerization of an aqueous monomer solution containing N-vinylcarboxylic acid amide and a water-soluble divinyl compound. Item 2. An electrophoresis gel according to Item 1.

The invention of claim 4 of the present invention is characterized in that the surface of the aqueous gel for electrophoresis which does not come into contact with the water-insoluble material exhibiting the gel bonding property is in contact with the plate-like rigid body having a concave notch at the top. The gel for electrophoresis according to any one of claims 1 to 3.

The invention according to claim 5 of the present invention is characterized in that the material of the plate-like rigid body having a concave cut in the upper part is glass or water-insoluble plastic. This is an electrophoresis gel.

According to a sixth aspect of the present invention, the aqueous gel for electrophoresis has a gel portion having a polymer concentration of 4.5% or less. Gel.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION A first limitation of the present invention is that one surface of an aqueous gel for electrophoresis having a well is in contact with a water-insoluble material exhibiting an adhesive property (gel bond property) to the aqueous gel for electrophoresis. The gel for electrophoresis is characterized in that:
A second limitation of the present invention is that, in the gel for electrophoresis according to claim 1, the aqueous gel for electrophoresis is produced by radical polymerization of a monomer aqueous solution containing acrylamide and a water-soluble divinyl compound. And A third limitation of the present invention is that, in the gel for electrophoresis according to claim 1, the aqueous gel for electrophoresis radically polymerizes an aqueous monomer solution containing N-vinylcarboxylic acid amide and a water-soluble divinyl compound. It is characterized by being manufactured. A fourth limitation of the present invention is that, in the gel for electrophoresis according to any one of claims 1 to 3, a surface of the aqueous gel for electrophoresis that is not in contact with a water-insoluble material having a gel bond property has a concave cut in an upper portion. Characterized by being in contact with a plate-like rigid body. A fifth limitation of the present invention is that in the gel for electrophoresis according to any one of claims 1 to 4, the material of the plate-like rigid body having a concave cut in the upper part is glass or water-insoluble plastic. I do. A sixth limitation of the present invention is the gel for electrophoresis according to any one of claims 1 to 5, wherein the aqueous gel for electrophoresis has a gel portion having a polymer concentration of 4.5% or less. .

In the present invention, the water-insoluble material having a gel bond property is referred to as a gel bond film used in a horizontal type (submarine type) electrophoresis method.
It is most convenient to use a PET film having a gel adhesive layer applied to one side. It is needless to say that not only a film but also a plate-shaped rigid body can be used as long as the gel adhesive layer is applied. Since the scope of application of the present invention defines a structure having a well for setting an analysis sample on the top of the gel, the gel is set upright, one side is set in contact with the electrode solution, and the gel is cooled by the contacted solution. Obviously, it is a vertical slab gel. In this method, the convection of the electrode solution used for cooling is smooth, and this method is particularly suitable for preventing temperature rise due to heat generation of the gel during energization.The analysis sample is set in a well isolated from other samples. Therefore, a clearer analysis can be performed than in the horizontal type electrophoresis. The wells are formed by polymerizing an aqueous monomer solution with a comb-shaped plastic called a comb set in a container called a plate. The polymerization of the monomer aqueous solution is triggered by radicals generated from a polymerization initiator such as a combination of ammonium persulfate (abbreviation: APS) and N, N, N ', N'-tetramethylethylenediamine (abbreviation: TEMED) or ultraviolet irradiation. Generally, radical polymerization is performed by radicals or the like.

Since the gel of the present invention is a vertical slab gel as described above, the upper well portion has a concave cut in the upper part of the container wall on the electrode (negative electrode) liquid side, and the aqueous gel and the electrode (negative electrode) ) It is necessary to be able to conduct electricity in the up and down direction by contact of the solution, and it is not realistic to immerse the rigid container wall with the concave cut in the staining solution from the viewpoint of sample arrangement. It is desirable that the container wall made of a water-insoluble material exhibiting a gel bond property has a simple rectangular shape. It is desirable that the material of the plate-shaped rigid body having a concave cut in the upper part is glass or a water-insoluble plastic such as PET. The monomer aqueous solution is polymerized in a sandwich container having a space in which a simple rectangular container wall exhibiting the gel bond property and a plate-shaped rigid container wall having a concave notch on the upper surface face each other and a spacer is filled with the monomer aqueous solution. By doing so, it is possible to produce an aqueous gel adhered to a water-insoluble material having one surface exhibiting a gel bond property. Needless to say, a comb is set in the container. The aqueous gel obtained in this manner, one surface of which is adhered to a water-insoluble material showing gel bond properties, even if the polymer concentration is low and fragile,
Rather than performing a dyeing operation without a soft, bare support like a normal vertical slab gel, the gel is reinforced with a water-insoluble material exhibiting gel bonding properties, so that the gel does not break during dyeing.

As the polymer forming the aqueous gel of the present invention, a conventionally known polyacrylamide-based polymer or poly-N-
Vinyl carboxylic acid amide gels can be used. Specifically, radical polymerization of an aqueous monomer solution (including a buffer) containing acrylamide and a water-soluble divinyl compound or an aqueous monomer solution (including a buffer) containing N-vinylcarboxylic acid amide and a water-soluble divinyl compound is used. It is produced by radical polymerization of As the water-soluble divinyl compound of the present invention, N, N-methylenebisacrylamide (abbreviation: BIS) is most frequently used, but a water-soluble divinyl compound such as N-allylacrylamide or N, N-diallyltartaramide is used. Can be. The aqueous monomer solution may contain a water-soluble polymer such as agarose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene oxide, polymethyl vinyl ether, or poly N-vinyl carboxylic acid amide in order to give the gel elasticity. it can. Generally, the gel for electrophoresis contains a buffer.
In the present invention, tris- (hydroxymethyl) aminomethane (abbreviation: TRIS), barbital, citric acid, or a buffer for isoelectric focusing can be contained in the aqueous gel. Further, if necessary, a surfactant such as sodium dodecyl sulfate (abbreviation: SDS), cetyltrimethylammonium bromide, polyethylene oxide sorbitan monooleate, etc. can be included. The present invention is particularly useful when the polymer concentration of the aqueous gel (monomer concentration at the time of preparation) is 4.5% or less, and is particularly useful in a low concentration part of a gradient gel or a part such as a stacking gel part. Even if there is a gel portion having a polymer concentration of 4.5% or less, the present invention is also applied to a case where the polymer concentration is 4.5% or less over the entire gel.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below with reference to embodiments, but the present invention is not limited only to the scope of the embodiments, but is constituted by the matters described in the claims of the patent. Needless to say, various changes can be made without departing from the scope of the technical idea to be performed.

(Gel Adjustment) A rectangular commercially available gel bond film having a width of 12 cm and a length of 10 cm was supported in close contact with a glass plate, and a glass spacer having a thickness of 1 mm was attached to a glass plate having a concave notch on the top. A monomer solution is injected by a syringe into a mold (container) in which the lower part of the liquid is sealed with silicone rubber, and finally, a PE comb is pushed from a concave notch to form a well, and pressed at 27 ° C. The polymerization was completed by allowing the mixture to stand for a period of time to prepare an electrophoresis gel (gel-1) used in the present invention. The composition of the monomer aqueous solution was 2.92% acrylamide monomer, BIS: 0.08%, TRIS: 0.4 mol (adjusted to pH 8.8 with HCl), SDS: 0.1
% And an appropriate amount of TEMED and APS were mixed and adjusted.

(Comparative gel preparation) width 12 cm, length 10 c
A 1 mm thick glass spacer is attached to a glass plate with a concave notch on the top of a rectangular glass plate with a thickness of m, and pressed with a syringe into a mold (container) sealed with a silicone rubber to seal the lower leak. A monomer aqueous solution was injected, and finally, a PE comb was pushed through a concave cut to form a well, and left standing at 27 ° C. for 3 hours to complete polymerization, and the gel for electrophoresis used in Comparative Examples of the present invention was used. (Comparative gel-1) was prepared. The composition of the aqueous monomer solution was 2.92% of acrylamide monomer, BIS: 0.
08%, TRIS: 0.4 M (pH 8.8 with HCl)
, SDS: 0.1% in aqueous solution containing TE
An appropriate amount of MED and APS were mixed and adjusted to the same composition as that used for preparing Gel-1.

(Electrophoresis Test) Gel-1 was set in a vertical electrophoresis apparatus manufactured by Atto Co., Ltd., and 5 μl of a protein sample was injected into each well to perform electrophoresis. The running buffer was 0.025 M TRIS, and 0.1
Contains 0.1% SDS in a 92M glycine-containing aqueous solution. As the protein sample, a commercially available HMW-SDS powder of a 2% distilled aqueous solution (BPB-colored) was used. Electrophoresis is 20 mA
Then, the energization was stopped when the electrophoresis end of BPB was 5 mm from the bottom.

(Dyeing) After the completion of the energization, the glass plate is removed,
The aqueous gel adhered to the gel bond film was immersed together with the gel bond film in a staining solution for 1 hour and in a decolorizing solution overnight (while updating to a new bath solution as appropriate). The composition of the staining solution is 0.05% Coomassie Brilliant Blue G-2.
50, 10% acetic acid, 30% methanol. The composition of the decolorizing solution is 12% acetic acid and 15% methanol. The aqueous gel was not damaged even after all the operations were completed, the electrophoresis image was clear, and it was suitable for drying and storing.

(Comparative Example) An electrophoresis test similar to that described above was performed using the comparative gel-1 in place of the gel-1 described above, and after finishing the energization, the glass plate was removed to dye an aqueous gel without a reinforcing material. When a spatula was put between the gel and the glass plate, the aqueous gel was broken, and the dyeing in the next step could not be performed.

[0021]

As described above, the aqueous gel adhered to the gel bond film of the present invention is handled together with the gel bond film to perform the dyeing and decoloring operations. It is possible to completely eliminate the breakage of the gel, which greatly contributes to the simplification of the analysis technique.

Claims (6)

[Claims] 1. An electrophoresis device for electrophoresis, wherein one surface of a planar aqueous gel for electrophoresis having a well is in contact with a water-insoluble material having an adhesive ability (gel bond property) to the aqueous gel for electrophoresis. gel. 2. The gel for electrophoresis according to claim 1, wherein the aqueous gel for electrophoresis is produced by radical polymerization of an aqueous monomer solution containing acrylamide and a water-soluble divinyl compound. 3. The electrophoresis aqueous gel according to claim 1, wherein the aqueous gel for electrophoresis is produced by radical polymerization of an aqueous solution of a monomer containing N-vinylcarboxylic acid amide and a water-soluble divinyl compound. gel. 4. The aqueous gel for electrophoresis according to claim 1, wherein the surface of the aqueous gel not in contact with the water-insoluble material exhibiting the gel bonding property is in contact with a plate-shaped rigid body having a concave notch on its upper part. 4. The gel for electrophoresis according to 3. 5. The gel for electrophoresis according to claim 1, wherein the material of the plate-shaped rigid body having a concave cut in the upper portion is glass or water-insoluble plastic. 6. The gel for electrophoresis according to claim 1, wherein the aqueous gel for electrophoresis has a gel portion having a polymer concentration of 4.5% or less.