JP3076200B2 - Electrophoresis gel, method for producing the same, and electrophoresis method - Google Patents

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, a method for producing the same, and an electrophoresis method. More specifically, in the fields of biochemistry and medicine, proteins, nucleic acids, etc. ranging from low molecular weight to high molecular weight are used. The present invention relates to a novel electrophoresis gel used for the separation and analysis of biological substances, and a method for producing the same and an electrophoresis method.

[0002]

2. Description of the Related Art Conventionally, natural materials such as paper, agarose, starch and the like have been used as a support for electrophoresis, but they have poor separation ability and bands are unclear. In recent years, gel electrophoresis using polyacrylamide as a support has been established, and high resolution can be expected due to the high-precision molecular sieving effect. Above all, the method using a polyacrylamide cross-linked product containing a buffer solution as the gel for electrophoresis has become the mainstream in the current gel electrophoresis method because the pore size of the gel can be adjusted arbitrarily depending on the acrylamide concentration and the composition of the cross-linking agent. , The protein to be separated,
A gel in which the concentration of acrylamide is changed according to the molecular weight of nucleic acids and the like is used for analysis. That is, generally, a high concentration polyacrylamide gel is used for separating proteins and nucleic acids having relatively low molecular weight, and a low concentration polyacrylamide gel is used for separating proteins and nucleic acids having relatively high molecular weight.

[0003] Regarding these techniques, for example, Japanese Patent Application Laid-Open
JP-A-3-502456, JP-A-62-91850, JP-A-2-22555 and the like. That is, JP-A-63-502456 discloses a method of supplying a buffer solution for electrophoretic separation. The method of supplying the buffer solution for the electrophoretic separation method is to make the electrode solution, which is usually used in a liquid state, into a gel to facilitate the contact with the separation gel, and to carry out the method using a support matrix in the form of a gel plate. A buffer solution is supplied to a horizontal electrophoresis separation method, in which two kinds of electrode buffers are mixed into one gel piece, and these gel pieces provide a separation area where a matrix between the gel pieces can be used. In contact with the support matrix. The gel piece is made of agarose or polyacrylamide. Furthermore, the content of the electrode buffer in each gel piece is 80-99.
% By weight.

Japanese Patent Application Laid-Open No. 62-91850 discloses a method for producing a polyacrylamide-based aqueous gel electrophoresis medium. In the method for producing the electrophoretic medium, the crosslinking initiation is carried out in the presence of an acrylamide monomer, a crosslinking agent, water and a polymerization initiating means. This method is a combination of light and does not contain a reducing agent.

[0005] Also, Japanese Patent Laid-Open No. 22555/1990 discloses that
A kit for an electrophoretic gel medium is disclosed. The electrophoresis gel medium kit is a kit for conveniently and safely forming a polyacrylamide-type polymer gel medium.

In order to separate proteins and nucleic acids having a wide range of molecular weight distribution, Japanese Patent Application Laid-Open No. 61-28512 discloses a technique relating to a gradient gel in which the acrylamide concentration is continuously changed in the direction of electrophoresis.
JP-A-63-210654, JP-A-1-2635
No. 48 publication.

That is, JP-A-61-28512 discloses a method for producing an electrophoretic gel substance. The method for producing the electrophoretic gel substance is carried out by homogenous photopolymerization using a water-soluble organic photoinitiator, or heterogeneous photopolymerization in which the organic initiator is suspended or dispersed in an aqueous photopolymerization solution. Beginning with a method of making a gradient gel with a continuous pore gradient.

[0008] JP-A-63-210654 and JP-A-1-263548 disclose an electrophoretic medium. That is, these publications describe a gel medium having an improved polyacrylamide-based aqueous gel concentration gradient having almost the same good high separation performance from a low molecular weight portion to a high molecular weight portion.

As a technique for producing an electrophoresis gel in which the concentration of acrylamide is changed stepwise, Japanese Patent Application Laid-Open No.
-302153. That is, a method for producing an electrophoresis gel obtained by sequentially superposing a plurality of aqueous monomer solutions having different acrylamide concentrations in a stepwise manner and then polymerizing the same is described.

[0010] When a protein is treated with sodium dodecyl sulfate (hereinafter abbreviated as SDS) and gel electrophoresis is performed with the charges of the protein molecules being equalized, separation is performed only by the difference in molecular weight. As tris (hydroxymethyl) aminomethane (hereinafter abbreviated as Tris)
A method of using a partially neutralized product of hydrochloric acid and a tris-glycine salt as an electrode solution for electrophoresis is described in Remley [UK Laemmli, Nature Vol. 227, 680 (197).
0)] for protein analysis. The gel buffer in this Lemley formulation has a pH of 8.
Approximately 10 to 20 mol% of Tris is partially neutralized with hydrochloric acid so as to give a value of 8.

As described above, polyacrylamide, N, N
-Acrylamide derivatives such as dimethylacrylamide,
Agarose and the like are generally used as a support for electrophoresis, but agarose has a disadvantage that it is brittle and has poor separation ability.On the other hand, in the case of polyacrylamide or the like, in the above-described known technique, polyacrylamide constituting a gel is used. The practical range of the concentration is 4 to 20% by weight. At a low concentration of 4% by weight or less, the gel strength is low, and at a high concentration of 20% by weight or more, there is a disadvantage that peeling from the container surface easily occurs. .

[0012]

It is known that high-molecular-weight proteins and nucleic acids can be rapidly separated by reducing the concentration of polyacrylamide or the like constituting a gel to a low concentration of 4% by weight or less. Acrylamide gels have low gel strength and cannot be used for separation of high molecular weight proteins, nucleic acids, and the like, and have the drawback that the measurable molecular weight range is narrow. An object of the present invention is to provide a novel electric power that can be used for the separation and analysis of biological substances such as proteins and nucleic acids ranging from low molecular weight to high molecular weight by remarkably expanding the measurable molecular weight range in the fields of biochemistry and medicine. An electrophoresis gel, a method for producing the same, and an electrophoresis method are provided.

[0013]

Means for Solving the Problems As a result of repeated studies to overcome the above problems, the present inventors have found that the above problems can be solved by using a gel containing a polymer electrolyte solution having a specific monomer unit as an aqueous solution. The present invention has been completed.

[0014] The first aspect of the present invention is an electrophoresis gel comprising, as a constituent, a gel containing an aqueous solution of a polymer having a monomer unit represented by the following general formula (I). .

Embedded image (However, R ₁ represents H or a methyl group)

According to a second aspect of the present invention, there is provided a method for producing an electrophoretic gel, comprising polymerizing an aqueous electrolyte of a water-soluble monovinyl monomer and a water-soluble divinyl monomer containing N-vinylcarboxylic acid amide as an essential component. Is the way.

The invention of claim 3 of the present invention is an electrophoresis method characterized in that the molecular sieve effect is adjusted by changing the copolymerization ratio of N-vinylcarboxylic acid amide and acrylamide.

[0017]

The gel for electrophoresis, the method for producing the same and the method for electrophoresis according to the present invention are constituted as described above, and operate as follows. A gel containing an aqueous electrolyte solution of a polymer having an N-vinylcarboxylic acid amide monomer unit as an essential component is not reduced in gel strength even when manufactured at a low polymer concentration of 4% by weight or less, and Even with the same polymer concentration, a gel with a larger pore size can be obtained compared to polyacrylamide gels, etc., so the transfer speed of high molecular weight proteins and nucleic acids etc. becomes faster, which is impossible with conventional polyacrylamide gels etc. The separation and analysis of high molecular weight proteins, nucleic acids, etc., became possible. It has been completely known that when an N-vinylcarboxylic acid amide monomer unit is used as an essential component, a gel having a larger pore size is obtained as compared with a case of a polyacrylamide gel or the like, and a moving speed of a high molecular weight protein or nucleic acid is increased. It has not been found and has been found for the first time by the present inventors.

A water-soluble monovinyl monomer containing N-vinylcarboxylic acid amide as an essential component and an appropriate amount of an aqueous electrolyte solution of a water-soluble divinyl monomer are copolymerized in accordance with a conventionally known conventional method to obtain the degree of crosslinking and pore size of the resulting gel. A controlled gel for electrophoresis can be produced.

Examples of the water-soluble divinyl monomer include N, N
-Methylenebisacrylamide is most often used, but other common divinyl compounds such as N, N-diallyltartaramide can also be used.

The aqueous monomer solution may contain a water-soluble polymer such as polyacrylamide, agarose, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene oxide, or polymethyl vinyl ether in order to impart elasticity to the gel and improve brittleness. it can.

The polymerization of the monomer is carried out by a polymerization initiator or a radical generated by irradiation with ultraviolet rays. As the polymerization initiator, a peroxide such as ammonium persulfate and N,
A redox-type polymerization initiator using a reducing agent such as N, N ', N'-tetramethylethylenediamine in combination is awarded, but is not particularly limited thereto. The peroxide and the reducing agent are used in an amount of 0.05 to 5 relative to all monomers.
% (W / v) is used. The polymerization temperature is not particularly limited as long as the initiator functions, but is usually 15 to
A range of 50 ° C. is preferred.

During the copolymerization, part of the N-vinylcarboxylic acid amide is replaced with acrylamide, and the pore size of the formed gel can be controlled by changing the copolymerization ratio of N-vinylcarboxylic acid amide and acrylamide. It is easy to control the molecular sieve effect. In the future, N-
There is a possibility that the pore size of the formed gel can be controlled without replacing part of the vinylcarboxylic acid amide with acrylamide.

[0023]

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

(Example 1) A polymer gel was produced under the following conditions using a monomer in which the mixing ratio (molar fraction) of acrylamide (abbreviated as AAM) and N-vinylformamide (abbreviated as NVF) was changed. An electrophoresis test was performed. Table 1 shows the relationship between the monovinyl monomer ratio (mixing molar ratio of AAM and NVF) and the mobility of a protein having a molecular weight of 94,000. (Test method and test conditions) A glass plate is assembled with a 1 mm thick spacer between a rectangular glass plate having a width of 12 cm and a length of 10 cm and a glass plate of the same size having a concave cut in the upper part. Monomer concentration 12.5% (%
T), N, N-methylenebisacrylamide (BIS)
Concentration 5% (% C), Tris concentration 0.09375 mol /
l, glycine concentration 0.192 mol / l, HCl concentration 0.07969 mol / l, SDS concentration 0.1% (W /
With respect to an aqueous monomer solution having the composition of V), 400 ppm of ammonium persulfate (APS) and N, N,
N ', N'-tetramethylethylenediamine (TEM
D) After adding and mixing 800 ppm, inject into the plate,
Polymerization was carried out by a conventional method to obtain a polymer gel for electrophoresis.

Using the polymer gel, a molecular weight of 94,00
No. 0 commercially available marker protein was subjected to electrophoresis, and the mobility (protein moving distance / dye moving distance) was measured. The marker protein was treated with β-mercaptoethanol and SDS, and added to 7% by weight of glycerin and 0.05% by weight of bromophenol blue (abbreviated as BPB) for the test. The composition of the buffer for electrophoresis was a Tris concentration of 0.02.
5 mol / l, glycine concentration 0.192 mol / l, S
This is a Remley prescription with a DS concentration of 0.1% (W / V). Electrophoresis was performed at a constant current of 20 mA, and the current was stopped when the BPB migration terminal was 5 mm from the bottom. Staining is 0.25
% (W / V) Coomassie Brilliant Blue-G250,
The reaction was performed for 2 hours in an aqueous solution containing 10% (V / V) acetic acid and 30% (V / V) methanol. Decolorization is 7%
The reaction was carried out day and night while appropriately changing to a new solution with an aqueous solution containing (V / V) acetic acid and 3% (V / V) methanol.

[0026]

Table 1 Molecular weight 94,000 at gel concentration of 12.5%
0 protein mobility

(Embodiment 2) In the same manner as in Embodiment 1, AA
Using a monomer in which the mixing ratio (molar fraction) of M and NVF was changed, a polymer gel was produced under the following conditions, and an electrophoresis test was performed. Monovinyl monomer ratio (AAM
Table 2 shows the relationship between the molar ratio of NBF and NVF) and the mobility of a protein having a molecular weight of 94,000. (Test method and test conditions) The monomer concentration was 7.5% (%
The test was performed under the same test method and test conditions as in Example 1 except that T) was used.

[0028]

Table 2 Molecular weight 94,000 at 7.5% gel concentration
Protein mobility

[0029]

The novel gel for electrophoresis of the present invention greatly expands the range of measurable molecular weights in the fields of biochemistry and medicine, and cannot be achieved with conventional polyacrylamide-based gels for electrophoresis. It can be used for separation and analysis of biological substances such as proteins and nucleic acids ranging from low molecular weight to high molecular weight. According to the production method of the present invention, it is possible to easily produce a gel for electrophoresis in which the degree of cross-linking and the pore size of the produced gel are controlled. By the electrophoresis method of the present invention,
The molecular sieve effect of the resulting gel can be easily adjusted.

────────────────────────────────────────────────── ─── Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 27/447 C09K 3/00 103 BIOSIS (DIALOG) CA (STN) JICST file (JOIS)

Claims (3)

(57) [Claims] 1. A gel for electrophoresis, comprising a gel containing an aqueous electrolyte solution of a polymer having a monomer unit represented by the following general formula (I) as a constituent element. Embedded image (However, R ₁ represents H or a methyl group) 2. A method for producing an electrophoresis gel, comprising polymerizing an aqueous electrolyte solution of a water-soluble monovinyl monomer and a water-soluble divinyl monomer containing N-vinylcarboxylic acid amide as an essential component. 3. An electrophoresis method comprising adjusting the molecular sieve effect by changing the copolymerization ratio of N-vinylcarboxylic acid amide and acrylamide.