JPS63262552A - Medium for electrophoresis - Google Patents

Abstract

PURPOSE:To obtain an gel medium showing highly accurate resolving power for analyzing a macromolecular component of a living body, by crosslinking and polymerizing a polymethacrylamide type copolymer represented by a specific formula and an acrylamide type compound in the presence of a water. CONSTITUTION:A polyacrylamide gel film is prepared by crosslinking and polymerizing a methacrylamide type copolymer having a constitutional repeating unit represented by formula I and an acrylamide type compound in the presence of water. As the acrylamide compound, an acrylamide homologue, a methacrylamide type compound or the like are designated and can be used alone or in a combination of two more kinds of them. The MW of the pref. used methacrylamide type copolymer is about 10,000-100,000 and the ratio thereof to the acrylamide compound is usually about 1-50wt.%, pref., about 5-40wt.%.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to an electrophoretic medium, and more particularly to an electrophoretic medium suitable for use in electrophoretic analysis of biopolymer components such as proteins. It is related to the medium for use.

[Background of the Invention] A typical embodiment of electrophoretic analysis is electrophoresis produced by coating or casting a film-forming material such as agar, cellulose, cellulose acetate, starch, silica gel, or polyacrylamide on a glass plate support. The membrane is impregnated with a buffer solution, the substance to be analyzed is attached onto it, and a voltage is applied to both ends of the support to develop (move) it on or within the support.
After that, the sample is dyed, and the optical density of the dyed sample is measured to perform quantitative analysis of each component of the substance.

For more information on electrophoretic analysis and electrophoretic membranes, please refer to "Experimental Electrophoresis Methods (Revised 5th Edition)"
” (Bunkodo, published in 1975), Aomizu and Nagai (eds.), “Latest Electrophoresis Methods” (Yokoyo Shoten, published in 1973), etc.

In recent years, electrophoresis has been frequently used to analyze biological components, and protein analysis in particular is frequently used in biochemical tests for disease diagnosis.

Filter paper has long been used as a membrane or sheet for electrophoresis, but as mentioned above, agarose membranes and polyacrylamide gel membranes have recently been used from a performance standpoint, and they have particularly effective molecular sieving effects. Many polyacrylamide gel membranes are currently in use.

The above polyacrylamide gel membrane is made by polymerizing and cross-linking monomers such as acrylamide with a bifunctional cross-linking agent such as N,N'-methylenebisacrylamide in the presence of a polymerization catalyst and in the absence of oxygen. It is obtained by letting

Note that an anionic surfactant may be added as a denaturing agent when producing a polyacrylamide gel membrane.

As mentioned above, since the polymerization reaction for forming polyacrylamide is radical crosslinking polymerization, the crosslinking polymerization is inhibited by the influence of oxygen. Therefore, polyacrylamide gel membranes must be manufactured in a state where oxygen is blocked. For this purpose, polyacrylamide gel membranes are generally used (cells made up of two glass plates) with a certain space, e.g. 0.3 mm.
It is manufactured by injecting a gel-forming liquid into a liquid (1 mm), blocking oxygen, and performing cross-linking polymerization to form a gel.

[Object of the invention] An electrophoresis medium with improved resolution suitable for use in electrophoretic analysis of biopolymer components is disclosed in Japanese Patent Application Laid-Open No. 1885-1985.
Although disclosed in Japanese Patent No. 3, in order to perform electrophoretic analysis of biopolymer components with even higher accuracy, it is desired to develop a gel medium that exhibits even higher resolution.

An object of the present invention is to provide a gel medium that exhibits high resolution and is used as an electrophoretic medium for electrophoretic analysis of biopolymer components.

[Summary of the Invention] The present invention provides a general formula (1) of methacrylamide copolymers having constituent repeating units represented by either of the following general formulas (1) and (2) [In the above formula, R11 is a hydrogen atom] or an alkyl group having 1 to 6 carbon atoms; R1'' Ql is -COO-1-CON-1 or an arylene group having 6 to 10 carbon atoms; Ll is -CO
A divalent group containing at least one O- or -〇〇N- bond and having 3 to 15 carbon atoms, or 10-1-N-1-CO-1-8O- 1 employee 5O2-1-SO, -1-S02N-1R” R
” R11 is a divalent group containing at least one bond of either -NCON-1 or -NCOO- and having 1 to 12 carbon atoms (however, Rit has the same meaning as above); R12 is - CH= CH2 or -CH2CH2x1 (where xl represents a group that can be substituted by a nucleophilic group or a group that can be eliminated in the form of HXI by a base); A1 is each monomer shown on the left side; is a divalent group derived from ethylenically unsaturated monomer copolymerizable with the unit; Xl, yl and zl represent molar percentages,
0 to 99, yl is 1 to 50, zl takes the remaining value, but zl may be O] General formula (2) [In the above formula, R21 is a hydrogen atom or 1 to 6 carbon atoms R22 is -CH=CH2 or -CH2CH2X2(
However, x2 represents a group that can be substituted by a nucleophilic group or a group that can be eliminated in the form of Hx2 by a base); L2 is an alkylene group having 1 to 6 carbon atoms,
arylene group with 6 to 12 carbon atoms, -CO
a group represented by Z2-, and a group represented by -COZ2R35- (where lli36 is an alkylene group having 1 to 6 carbon atoms, or an arylene group having 6 to 12 carbon atoms, and Z2 is an oxygen atom or NH); A2 is a divalent group derived from an ethylenically unsaturated monomer copolymerizable with one unit of each monomer shown on the left side; Yes; x2, y2 and z2 represent mole percentage, x2 is 5
0 to 99, y2 is 1 to 50, z2 takes the residual value, however, Z2 may be O. Polyacrylamide-based aqueous gel electrolytic formed by cross-linking polymerization of j and an acrylamide-based compound in the presence of water. It provides a migration medium.

The electrophoretic medium of the present invention exhibits high resolution.

In addition, it has good processability, which means that cutting the polyacrylamide gel membrane with a knife such as cutting a long electrophoresis medium or adding sample slots can cause damage to the gel membrane or change in shape near the cut surface. Since the method can be easily and reliably carried out without any complication, the resulting electrophoresis medium exhibits excellent performance and can be easily manufactured in mass production.

When mass producing the electrophoresis medium, it is advantageous to form the medium on a support made of plastic material. Such a shape facilitates mass production of the electrophoresis medium of the present invention, and since the support is lightweight and hard to break, the electrophoresis medium can be easily stored, transported, and the like. Therefore, polyacrylamide gel membrane (
This method is extremely effective in realizing a gel membrane production/use system that centrally produces gel membranes (electrophoresis media) and supplies them to electrophoresis operators as needed.

[Detailed Description of the Invention] The support for the electrophoresis medium of the present invention includes a glass plate,
Any water-resistant sheet-like support can be used, such as polymer-coated paper, sheets of plastic material, and the like. However, in order to take advantage of the electrophoresis medium of the present invention,
Preferably, a sheet of plastic material is used as the support.

Various plastic sheets can be used as the plastic sheet support for the electrophoresis medium. Preferred examples of plastic sheets include wood-philic polymers or polymers whose surfaces have been made hydrophilic by known surface treatments (
Examples: polyethylene terephthalate, polycarbonate of bisphenol A, polyvinyl chloride, vinylidene chloride O
Vinyl chloride copolymer, polymethyl methacrylate,
Examples include sheets (including films and plate-like materials) of polyethylene, polypropylene, cellulose acetates, cellulose acetate propionate, etc.).

A particularly preferred support is polyethylene terephthalate.

Next, the electrophoresis medium layer (hereinafter also referred to as gel medium layer, polyacrylamide gel membrane, or simply gel membrane) will be explained.

As mentioned above, polyacrylamide gel membranes are generally produced by dissolving or dispersing an acrylamide compound and a crosslinking agent in water as an aqueous solution or dispersion to prepare a gel forming solution, and then crosslinking and polymerizing both in the solution. It can be obtained by cross-linking and polymerizing to form an aqueous gel film. In this specification, unless otherwise specified, the term ``dissolution (in water)'' includes both dissolution (in water) and dispersion (in water), and the term ``dissolution (in water)'' includes both an aqueous solution and an aqueous dispersion. It is called an aqueous solution. In addition, as a solvent or dispersion medium,
Also included are mixtures of organic solvents and water, which may be added as desired.

On the other hand, in the present invention, a polyacrylamide gel membrane is produced by crosslinking and polymerizing a specific polymethacrylamide copolymer and an acrylamide compound in the presence of water. Note that when a polyfunctional crosslinking agent (particularly a trifunctional crosslinking agent) is involved in carrying out this crosslinking polymerization, the resolution and processability of the resulting polyacrylamide gel film are further improved.

Examples of acrylamide compounds that can be used in the present invention include acrylamide, acrylamide homologues such as N-methylacrylamide, N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide, and diacetone acrylamide, and methacrylamide compounds. Examples include methacrylamide, and these compounds can be used alone or in combination of two or more. Among these acrylamide compounds, acrylamide is most preferred, and a combination of acrylamide and one or more other acrylamide compounds is also preferred.

The specific polymethacrylamide copolymer involved in the production of the polyacrylamide gel membrane of the present invention is a methacrylamide copolymer having a structural repeating unit represented by either of the following general formulas (1) and (2).

In the above formula, R11 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, examples of which include methyl, ethyl, butyl, n-hexyl, and the like.

R'' ■ Ql is -COO-1-CON-1 or an arylene group having 6 to 10 carbon atoms, examples of which include the following groups.

H3 -COO-5-CONH-5-CON-1Ll is -C
・A divalent group containing at least one bond of 00- or -〇〇N- and having 3 to 15 carbon atoms, or -0-1-N-1-CO-1-SO −
A divalent group containing at least one of 1-5O2-5-SO, -1-3O2N-1 and having 1 to 12 carbon atoms (R11 has the same meaning as above), examples thereof includes the following groups: Note that the bonding direction of these divalent groups in general formula (I) can be in any direction as long as the bonding between Ql and S02 is possible.

-CH2COOCH2- CH2COOCH2CH2- CH2CH2COOCH2- +CH2COOCH2CH2- +CH2C00CH2CH2- -CH2NHCOCH2- CH2N HCOCH2CH2- f CH2NHcOcH2CH 2-+ CH2 Chi 6 N HCOCH2CH2-MoCH2 soup NHCO
CH2CH2- -CH20CH2- -CH2CH20CH2CH2CH2- -COCH2C
H2- -CH2COCH2CH2- -5OCR2CH2- -CH25OCH2CH2- -SO2CH2CH2- -5O2CH2CH2SO2CH2CH2-O -3O, CH2C00CH2CH2- S Os CH2CH2COOCH2CH2-3O2
N HCH2COOCH2CH2--SO2NHCH2
CH2C00CH2CH2-N HCON HCH2C
H2- CH2N HCON HCH2CH2-N HCOOC
H2CH2- -CH2NHCOOCH2CH2- R12 is either -CH=CH2 or -CH2CH2Br (where xl represents a group that can be substituted by a nucleophilic group or a group that can be removed by a base in the form of HX''), and this Examples of Xl include the following groups:

Halogen atoms (chlorine, bromine, etc.): Hydroxyl group; Alkylsulfonyloxy (e.g., methylsulfonyloxy H3cms-o-, ethylsulfonyloxy, propylsulfonyloxy, etc.); Arylsulfonyloxy (
(e.g., phenylsulfonyloxy); alkylcarbonyloxy (e.g., acetoxy, propionyloxy,
(trifluoromethylcarbonyloxy, dichloromethylcarbonyloxy, etc.) Therefore, examples of R12 include the following groups.

-CH=CH2, -CH2CH2C statement, -CH2CH2
Br, l -CH2CH2-O-S-CH3, CH2CH20Hl -CH2CH200CCH3, CH2CH200CCF 3, -CH2CH200CCC 3゜A1 is derived from an ethylenically unsaturated monomer copolymerizable with one unit of each monomer shown on the left side. Examples thereof include the following groups derived from ethylenically unsaturated radicals.

Ethylene, propylene, l-butene, isobutyne, styrene, chloromethylstyrene, hydroxymethylstyrene, sodium vinylbenzenesulfonate, sodium vinylbenzylsulfonate, N,N,N-)rifthyl-
N-vinylbenzylammonium chloride, N,N-dimethyl-N-benzyl-N-vinylbenzylammonium chloride, α-methylstyrene, vinyltoluene, 4
-vinylpyridine, 2-vinylpyridine, benzylvinylpyridinium chloride, N-vinylacetamide, N
- vinylpyrrolidone, 1-vinyl-2-methylimidazole, monoethylenically unsaturated esters of aliphatic acids (e.g. vinyl acetate, allyl acetate), ethylenically unsaturated mono- or dicarboxylic acids and their salts (e.g. acrylic acid) , methacrylic acid, itaconic acid, maleic acid,
sodium acrylate, potassium acrylate, sodium methacrylate), maleic anhydride, esters of ethylenically unsaturated monocarboxylic or dicarboxylic acids (e.g. n-butyl acrylate, n-hexyl acrylate, hydroxyethyl acrylate, cyanethyl acrylate) , (diethylamino)ethyl acrylate, methyl methacrylate, n-butyl methacrylate, benzyl methacrylate, hydroxyethyl methacrylate, chloroethyl methacrylate, methoxyethyl methacrylate, (diethylamino)ethyl methacrylate,
N,N,N-triethyl-N-methacryloyloxyethylammonium p-)luenesulfonate,
N,N-diethyl-N-methyl-N-methacryloyloxyethylammonium p-toluenesulfonate, dimethyl itaconate, maleic acid monobenzyl ester)
, amides of ethylenically unsaturated monocarboxylic or dicarboxylic acids (e.g. N,N-dimethylacrylamide,
N-methylolacrylamide, N-[(dimethylamino)propyl]acrylamide, N,N,N-1 remethyl-N-(acryloylpropyl)ammonium, P-
1 luenesulfonate, 2-, sodium acrylamide-2-methylpropanesulfonate, acryloylmorpholine, methacrylamide, N,N-dimethyl-N"-
Acryloylpropanediamine probionate betaine, N,N-dimethyl-N'-methacryloylpropanediamine acetate betaine.

When the copolymer of general formula (1) is used as a crosslinked latex, A1 contains at least two copolymerizable ethylenically unsaturated groups in addition to the groups derived from the ethylenically unsaturated monomers mentioned above. Seven Tsuma with more than (
For example, groups derived from divinylbenzene, methylene bisacrylamide, ethylene glycol diacrylate, trimethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylene glycol dimethacrylate, neopentyl glycol dimethacrylate, etc.) can be used.

xl, yl and zl represent mole percentage, xl is 5
0 to 99, and yl takes a residual value of 1 to 50.21, but zl may be O.

In the above formula, R21, R22 and A2 have the same meanings as R11, RL2 and A1 in the general formula (1), respectively, and the examples thereof are also the same.

L2 is an alkylene group having 6 carbon atoms from 1 (
(e.g., methylene, ethylene, imbutylene, etc.), arylene groups having 6 to 12 carbon atoms (e.g., phenylene, tolylene, naphthalene, etc.), groups represented by -coz2-, and groups represented by -COZ2R"- (However, R06 is an alkylene group having 1 to 6 (II) carbon atoms, or an arylene group having 6 to 12 carbon atoms, and Z2 is an oxygen atom or N
is a divalent group selected from H).

x2, y2 and z2 represent mole percentage, x2 is 5
0 to 99, y2 takes the value of 1 to 50, and z2 takes the remaining value, however, z2 may be O.

Typical vinylsulfonyl group that can be used to synthesize a polymer having a constituent repeating unit represented by either of the above general formulas (1) or (2) or an ethylenically unsaturated functional group that is a precursor thereof The method of synthesizing the monomer is specifically described in JP-A-61-18853.

Preferred methacrylamide copolymers used in the present invention are those represented by the following formula.

Here, x, y, and z are molar percentages, and R and R' represent the following substituents.

A-1x=92, Y=8, z=O R= Coo CH2CH2000CH2CH25o□
CH=CH2A-2x=90.7=10. z=0 R” GONHCH2NHGOGH2CH2SO2CN
= 002A-3x=80. y=8, z=12 R= C0NHCH2NHGOGH2CH2So 2
C) l= OH2R″= C0NH2C(OH,) 2
0H2COGH.

A-6x=92, y=8, z=O n A-7x=92, Y=8, 2=0 R= C0NHCH2NIC:OGH2CH2So□0
H2C) I2CIA-8x=80. y=8, z=12 R= C0NHGH2NHCOGH2Cn2so20H
2CH2CIR” = C0NH2C(C)Is)
2 C1(2CDC)i 3R' = GO
NHGH20H R' = C0N(CH3)2 A-12x = 92, y=8. z=0R” Goo
CH2CH20COCH2SO2CH=CH2 By the way, a polymer using an acrylamide component in place of the methacrylamide component of the present invention was filed in Japanese Patent Application Laid-Open No. 18853/1983. Specifically, here, X, y, and z are mole percentages, and R and R' represent the following substituents.

B-1x=92, y=81, z=Q R= 000 CH2CH2000CI2 CH2SO
2GH= GH2B-2x=90, y=10.2=0 R-0014)1(l[2NHC:0CH2012SO
2GH=CH2B-3x=80. y=8, z=12 R= C0NHGH2NHCOC:H2co 2SO2
cn=CH2R'=GONH2G(CH3) 2
0H2GOCHsB-4x=92, y-=8.2=0 B-6x=92, V=8, z=0 υ B-7x=92, V=8, 2=0 R= GONHI:H2NHCOCH2GH2SO2C
H2CH2CIB-8x=80, V=8, z=
12R= C0NHCH2NHCOCH2CH2SO2
CI2 CH2CIR' = C0N) 120CGHs
) 2 G) 12 GOCI*R': c
oNncH20H B-11x = 80, V=8, z=12R'
= C0W(OH:+) 2B-12x =
92, y=8, z=0R= Coo CH20
H20000H2SO2C)I=CH2 Next, synthesis examples of copolymers named A-2 and A-8 among the above copolymers will be shown.

[Synthesis Example 1] Synthesis of copoly-N-([3-(vinylsulfonyl)propanamidocomethyl)acrylamide-methacrylamide (A-2) In a 200 mJ reaction vessel, N-[-3-(2-chloroethyl 5.65 g of methylcoacrylamide (sulfonyl)propanamide, 15.3 g of methacrylamide and 80 g of 50% methanol aqueous solution
Add m-mon and heat to 60℃ while stirring, 2゜2'-
Azobis(2,4-dimethylvaleronitrile) 0.1g
After 30 minutes, 0.1 g of the same substance was added, and the mixture was heated and stirred for 2 hours.

Thereafter, the mixture was cooled to about 10°C with ice water, and 2.5 g of triethylamine dissolved in 80 mjQ of methanol was added, stirring was continued for 2 hours, and the reaction solution was poured into 1 m of acetone with stirring to generate The resulting precipitate was collected by filtration to obtain 16.3 g of a white polymer (corresponding to A-2 above) (yield: 80%). The sulfonyl group content of this polymer was 0.90×10 −3 equivalents/g.

[Synthesis Example 2] Copoly-N-([3-(2-chloroethylsulfonyl)propanamidocomethyl)acrylamide-methacrylamide-N-(1,1-dimethyl-3-
Synthesis of oxobutyl)acrylamide (A-8) In a 500ml reaction vessel, 3g of N-[3-(2-chloroethylsulfonyl)propanamidomethylcoacrylamide lo, 35.0g of methacrylamide, N-(1,
11.3 g of l-dimethyl-3-oxobutyl)acrylamide (diacetonamide) and 160 m of a 50% aqueous methanol solution were added, and heated to 60°C with stirring.
Add 0.2 g of 2゜2゛-azobis=(2,4-dimethylvaleronitrile), and after 1 hour add another 0.2 g of the same.
In addition, heating and stirring were continued for 2 hours. The reaction solution was put into a cellulose tube, dialyzed for 2 days, and lyophilized to 45.0
A white polymer (corresponding to A-8 above) of g was obtained. (Yield: 80%) The chloroethylsulfonyl group content of this polymer was 0.8 x 10-3 equivalent/g.

The molecular weight of the methacrylamide copolymer preferably used in the present invention is in the range of about 10,000 to about 100,000.

The ratio of the acrylamide compound and methacrylamide copolymer used in the crosslinking polymerization reaction is usually about 1 to 50% by weight, preferably about 5 to 40% by weight, based on the acrylamide compound.

As mentioned above, a crosslinking agent may be used in conjunction with the production of the polyacrylamide gel membrane of the present invention. As a crosslinking agent,
For example, r E]ectrophoregigJ19
Known compounds described in 81.2.213-228 and the like can be used. Examples of crosslinking agents include N,N'-methylenebisacrylamide F (BIS);

No-propylene bisacrylamide (PBA); di(
Acrylamide dimethyl) ether (DAE): 1,2
-diacrylamide ethylene glycol (DEG); ethylene urea doth acrylamide (EUB); ethylene diacrylate (EDA); N,N'-diallytartardiamide (N,N'-dially
ltartardiamide: DA
T D ); N,N''-bisacrylylcystamine (N,N'-bisacrylylcystamine)
difunctional compounds such as amine, B A C), and triallyl cyanurate, triallyl in cyanurate, 1,3.5-)lyacryloylhexahydro-s-
Examples include trifunctional compounds such as triazines. The crosslinking agents may be used alone or in combination of two or more.

The crosslinking agent is used in an amount ranging from about 30% by weight or less, preferably about 10% by weight or less, based on the total weight of the acrylamide compound, methacrylamide copolymer, and crosslinking agent.

The gel concentration of the polyacrylamide gel used in the production of the electrophoresis medium of the present invention is S, Hjer
ten: r Arcb, Biochem, Bio
phys, Jl (5upp1.), 147 (19
62), an acrylamide compound, a methacrylamide copolymer, and water,
and optionally a crosslinking agent, the amount of acrylamide-based compound and methacrylamide-based copolymer (and optionally crosslinking agent) is about 3
A range of w/v% to about 30 w/v% is preferably used.

The electrophoresis medium of the present invention is mainly used to advantageously analyze proteins or complex proteins (e.g., lipoproteins, glycoproteins, etc.), and the electrophoresis medium layer contains an anionic interface as a denaturing agent. Activators can be included.

When the analysis sample is a protein or complex protein (eg, lipoprotein, glycoprotein, etc.), it is often preferable or necessary to include an anionic surfactant.

However, it goes without saying that the anionic surfactant may not be included in the electrophoresis medium layer. For example, an electrophoresis medium layer that does not contain an anionic surfactant can be used for purposes such as genetic disease diagnosis based on DNA fragment analysis or DNA structure analysis using restriction enzyme digestion.

By incorporating an anionic surfactant into the electrophoresis medium layer, it becomes possible to efficiently separate proteins or complex proteins and measure their molecular weights.

Examples of anionic surfactants include alkyl sulfates, and alkyl sulfates having a long chain alkyl group having 10 or more carbon atoms are particularly preferably used. As cations that form salts, alkali metal ions such as sodium ions, potassium ions, and lithium ions are generally used, and among these, sodium ions are easily used. Among alkyl sulfates, dodecyl sulfate (
(sodium salt, potassium salt, lithium salt, etc.) are preferred, and among these, sodium dodecyl sulfate (SDS) is most preferred. By incorporating SDS into the electrophoresis medium layer of the present invention, it becomes possible to efficiently separate proteins or complex proteins and measure their molecular weights.

The content of the anionic surfactant as a denaturing agent ranges from about 0-05 w/v% to about 2 Ow/v with respect to the gel-forming solution.
v%, preferably from about 0,1 w/V% to about 1.5
The range is w/v%.

A water-soluble polymer is optionally added to the electrophoretic medium layer of the present invention. As the water-soluble polymer, addition polymerization type or condensation polymerization type water-soluble polymer can be used. Examples of addition polymers include nonionic water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide. Examples of polycondensation polymers include polyethylene glycol,
Examples include nonionic water-soluble polyalkylene glycols such as polypropylene glycol. Among these water-soluble polymers, polyacrylamide and polyethylene glycol are preferred.

The molecular weight of the water-soluble polymer is preferably in the range of about 10,000 to about 1,000,000. The water-soluble polymer is used in an amount ranging from about 2% to about 100%, and preferably from about 5% to about 50%, based on the total weight of monomer and crosslinker.

The addition of a water-soluble polymer makes the polyacrylamide gel film plastic, which prevents it from breaking during cutting, and the gel film also has plasticity when drying, which has the advantage of improving brittleness and making it less likely to break. be. Furthermore, the viscosity of the gel film can be controlled by selecting the molecular weight and amount of the water-soluble polymer added.

It is preferable that the polyacrylamide gel membrane of the present invention further contains agarose. Any agarose can be used, and any of low electroosmotic, medium electroosmotic, and high electroosmotic agaroses can be used. Examples of agarose that can be used include JP-A-55-15730 and JP-A-55-110946.
Agarose and the like are disclosed in various publications such as Japanese Patent Publication No. 57-502098. The agarose is about 0.2% w/v to about 2% by weight based on the volume of the aqueous gel containing the monomer and crosslinker.
w/v%, preferably from about 0.3 w/v% to about 1.2 w/v%.

When the polyacrylamide gel film contains agarose, it is possible to control the viscosity of the gel forming solution to an appropriate level by changing the temperature of the gel forming solution, which makes it possible to stop its fluidity and to prevent the gel film from forming. There is an advantage that molding becomes easier during the molding operation.

The electrophoresis medium layer of the present invention can contain a pH buffer. The buffer can be appropriately selected from known buffers capable of buffering to a pH value within the range of pH 2.5 to 1000, depending on the sample to be electrophoretically analyzed.

As a buffering agent that can be used, Nippon Kagaku Gosen "Chemical Handbook Basic Edition" (Tokyo, Marukubi, published in 1966) 1312-13
Page 20; “Latest Electrophoresis Methods” edited by Aomizu and Nagai (Tokyo, Tokonyo Shoten, published in 1973, pages 320-322;
r Data for Bioche+wi
calRe5earchJ (R,M.

C. Dawson at al, ed., 2nd edition, Oxfo
rd at theGlarendon Press,
Published in 1969) pp. 476-508; r Bio
chemistry 4 5.467 (1966),
rAnalytical Biochemistry
J 104.300-310 (1980) and the like.

Examples of buffers include barbital-containing buffers, tris(hydroxymethyl)aminomethane (Tris)-containing buffers, phosphate-containing buffers, borate-containing buffers, acetic acid or acetate-containing buffers. agent, buffer containing citric acid or citrate, buffer containing lactic acid or lactate, buffer containing glycine, N,N-bis(2-hydroxyethyl)glycine (Bicine), N-2-hydroxy Ethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid (HEPPSO) or its salt, N-
2-Hydroxyethylpiperazine-N”-3-propanesulfonic acid (EPPS) or its salt, N-[)
Examples include lith(hydroxymethyl)]-]3-aminopropanesulfonic acid TAP'S) or a salt thereof, and acids, alkalis, or salts that may be combined with any of these as necessary. Specific examples of preferred buffers include potassium dihydrogen phosphate/disodium hydrogen phosphate, Tris/sodium borate, and Tris/sodium borate.
Sodium borate/EDTA2Na salt, Tris/citric acid, Parbital sodium-sodium acetate, Parbital sodium/hydrochloric acid.

Barbital/parbital sodium, acetic acid/sodium acetate, lactic acid/sodium lactate, citric acid-disodium hydrogen phosphate, Bicine, HEPPSO
, HEPPSO sodium salt, EPPS, EPPS sodium salt, TAPS, TAPS sodium salt, and the like.

Further, the electrophoresis medium layer of the present invention may contain a polyol compound such as glycerin or ethylene glycol as a wetting agent. The content of the polyol compound is selected from the range of about 0.1 w/v% to about 40 w/v% based on the volume of the electrophoretic medium layer. Among these compounds, glycerin is particularly preferred. By adding a wetting agent, it is possible to prevent polyacrylamide gel membranes from drying out due to extreme water evaporation during storage, and also to prevent brittleness caused by extreme dryness and prevent cracking of gel membranes. There is an advantage that physical properties are improved.

The electrophoresis medium layer of the present invention may contain an antioxidant, if necessary. As the antioxidant, various compounds known to be able to be incorporated into polyacrylamide gel membranes can be used. Examples of antioxidants include dithiothreitol, 2-mercaptoethanol, and the like.

The electrophoresis medium layer (polyacrylamide gel membrane) in the electrophoresis medium of the present invention is typically made of a monomer represented by acrylamide, a difunctional allyl compound, or an acryl compound as described above. It is obtained by radical crosslinking polymerization of a compound (crosslinking agent), water-soluble polymer, agarose, etc. with an acrylamide-based compound and a methacrylamide-based copolymer (and optionally a crosslinking agent) in a substantially uniform aqueous solution. ,
The water-soluble polymer and agarose are substantially dispersed in a three-dimensional cross-linked polymer formed from an acrylamide-based compound and a methacrylamide-based copolymer (and optionally a cross-linking agent), so that the polymer chains of the latter two and the three-dimensional cross-linked polymer It is estimated that the structure has an intertwined structure.

The radical crosslinking polymerization reaction used in the present invention can be carried out in the absence of molecular oxygen in the presence of peroxide and/or by known methods such as ultraviolet irradiation. moreover,
This crosslinking polymerization reaction can also be accelerated by heating or ultraviolet irradiation.

As a catalyst for radical crosslinking polymerization, rElectr.

phoresisJ 1981.2.213-219.
19B1.2.220-228: The low-temperature radical polymerization initiators can be appropriately selected and used from among the known low-temperature radical polymerization initiators described in "Latest Electrophoresis Methods J" (published in 1973), edited by Aomizu and Nagai. Preferred radical polymerization Specific examples of initiators include β-dimethylaminopropionitrile (DMA
PN)・ammonium peroxonisulfate mixture, N,N
, N', N'-tetramethylethylenediamine (TE
MED)・Ammonium peroxonisulfate mixture, TE
Examples include combinations of MED/riboflavin mixtures, TEMED/riboflavin/hydrogen peroxide mixtures, and ultraviolet irradiation. The content of the radical polymerization initiator is approximately 0.3% by weight based on the total weight of the acrylamide compound and methacrylamide copolymer (and optionally crosslinking agent).
from about 5% by weight, and preferably from about 0.5% to about 3% by weight.

The polyacrylamide gel membrane of the present invention is produced by coating a gel-forming liquid on a support having a smooth surface by a known method to form a coated layer, and then subjecting the coated layer to crosslinking polymerization. However, when applying the gel-forming liquid to the support, in order to improve the adhesion between the electrophoresis medium layer and the support, an adhesive layer is provided on the surface of the support in advance, and the gel-forming liquid is applied onto this. However, a gelling method can also be used. The application of an adhesive layer is particularly advantageous when plastic carrier sheets are used.

When cross-linking and polymerizing the gel-forming liquid on the surface of the support,
The gel-forming liquid can be further covered with a covering material such as a cover film, sheet or plate. A cover film or the like used for this purpose may be made of the same material as the support. The thickness of this coating material is 300 ILm or less, with a practically preferred range of about 4 to 2001 Lm.

In addition, when producing the electrophoretic medium of the present invention, it is also possible to use a method in which the electrophoretic medium is formed on a cover film or the like, and then a support is attached thereon.

The electrophoretic medium of the present invention can be prepared by horizontal and vertical plate electrophoresis,
It can be used for any method such as disk electrophoresis.

An electrophoretic medium to which a cover film is attached during the production of the electrophoretic medium layer is subjected to electrophoresis, and the cover film can be removed after electrophoresis, or the electrophoresis result can be processed by autoradiography without removing the cover film. Analysis is performed.

Next, examples of the present invention will be shown.

[Example 1 and Comparative Example 1] Polyethylene terephthalate (polyethylene terephthalate) having a thickness of 180 ILm and having a hydrophilic surface (PET) sheet (supporting body) was prepared as described in Table 1 to a thickness of about 300 pm. A polyacrylamide gel membrane was manufactured by applying a gel-forming solution and cross-linking polymerized in a nitrogen atmosphere.

A sample injection port was formed in this using a cutter. Next, a PET sheet (cover film, thickness 100 gm) with a hydrophilic surface was placed on the surface of this gel membrane to cover it, thereby obtaining an electrophoretic medium material.

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Standard proteins were electrophoresed using the electrophoresis media materials described above. This electrophoresis medium was then mixed with 0.1% Goomassie Blue.
ue) R-250 (C, I, Con5tituti
on No. 42660) The sample was immersed in a staining solution for staining, and the separable portion of the protein was visually examined. The separation patterns that appeared on the gel membranes of sample numbers 1 and 2 (samples according to the present invention) showed sharp bands compared to the separation patterns that appeared on the gel membranes of sample numbers 3 and 5 (comparative samples). .

[Example 2 and Comparative Example 2] An electrophoretic medium material was formed in the same manner as in Example 1 using the gel forming solution shown in Table 2.

Standard proteins were subjected to electrophoresis using the electrophoresis medium materials described above. This electrophoresis medium was then coated with 0.1% Coomassie Blue R-
250 (C, I, Condition No.
42660) The sample was immersed in a staining solution for staining, and the separable portion of the protein was visually examined. The separation pattern that appeared on the gel membrane of Sample No. 8 (sample according to the present invention) showed sharp bands compared to the separation pattern that appeared on the gel membranes of Sample Nos. 9 to 12 (comparative samples). moreover,
In the separation patterns that appeared on the gel film of sample numbers 6 to 7 (samples according to the present invention), the band sharpness was improved compared to the separation pattern that appeared on the gel film of sample number 8 above. .

[Example 3 and Comparative Example 3] 4.5 g of acrylamide, 1,3,5-triacryloylhexahydro-S-triazine o, to
g, copoly-N-([3-(vinylsulfonyl)propanamidocomethyl)acrylamide-methacrylamide (A-2) 1.2g, polyacrylamide 0.6g,
Agarose (low electro-osmolarity, gelling temperature 36℃) 0.3
g,) lis(hydroxymethyl)aminomethy [CA
S Registry No.7? -86-1] 1
, 08g, boric acid 0.55g, EDTA・2Na salt 9
3 mg, and 100 mJ containing 20 g of glycerin.
To an aqueous solution of L, 1.3 ml of ammonium beloxodisulfate (5 wt% aqueous solution) as a polymerization initiator and TEM
A gel forming solution containing 33 gM of ED was applied to a thickness of 1 mm, and this was cross-linked and polymerized to obtain a polyacrylamide gel film. A sample injection port was formed in this using a cutter. Next, a PET sheet (cover film, thickness 100p, m) with a hydrophilic surface was placed on the surface of this gel film with the woodphilic surface facing down to cover it, thereby obtaining a medium material for electrophoresis. Ta.

As a comparative sample, 4.5 g of acrylamide and 0.10 g of 1.3.5-)lyacryloylhexahydro-5-triazine were placed on the surface of a PET sheet that had been similarly treated.
, copoly-N ([3-(pyrylsulfonyl)propanamidocomethyl)acrylamide-methacrylamide (B-2) 1.2g polyacrylamide 0.6g, agarose (low electroosmoticity, gelation temperature 36°C) 0 .3g,
Tris(hydroxymethyl)aminomethane 1.08g,
Boric acid 0.55g, EDTA・2Na salt 93mg,
1.3 mJ1 of ammonium belloxonisulfate (5 wt% aqueous solution) as a polymerization initiator and TEMED33p-
A gel-forming solution to which 1 was added was applied to a thickness of 1 mm, and this was cross-linked and polymerized to obtain a polyacrylamide gel film for comparison. A sample injection port was formed in this using a cutter. Next, a PET sheet (cover film, thickness 100 gm) with a hydrophilic surface was placed on the surface of this gel membrane with the wood-philic surface facing down to cover it, to obtain a comparative electrophoresis medium material. Ta.

E. coli plasmid pBR-322 was transformed with restriction enzyme Asul.
The treated samples to be analyzed were electrophoresed on the above gel membrane, and the DNA degradation patterns were compared using ethidium bromide staining. The separation pattern that appeared on the gel membrane of the former sample according to the present invention showed sharp bands compared to the separation pattern that appeared on the gel membrane of the comparative sample.

This result means that when electrophoresis is performed using the electrophoresis medium according to the present invention, the bands of the separation pattern obtained are sharp, making identification and fractionation easy and highly accurate. ing.

Claims (1)

[Claims] 1. A methacrylamide copolymer having a constituent repeating unit represented by either of the following general formulas (1) and (2): General formula (1) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [ In the above formula, R^1^1 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Q^1 is -COO-, ▲ has a mathematical formula, chemical formula, table, etc. ▼, or 6 to is an arylene group having 10 carbon atoms; L^1 contains at least one bond of -COO- or Divalent groups with -O-, ▲numerical formulas, chemical formulas, tables, etc.▼
, -CO-, -SO-, -SO_2-, -SO_3-, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, or ▲Mathematical formulas,
There are chemical formulas, tables, etc. A divalent group containing at least one of the bonds ▼ and having 1 to 12 carbon atoms (R^1^1 has the same meaning as above); R^ 1^2 is -CH=CH_2 or -CH_2CH_2
X^1 (where X^1 represents a group that can be substituted by a nucleophilic group or a group that can be eliminated in the form of HX^1 by a base); A^1 is shown on the left side. is a divalent group derived from an ethylenically unsaturated monomer copolymerizable with each monomer unit; x^1, y^1 and z^1 represent a molar percentage, x
^1 is 50 to 99, y^1 is 1 to 50, z^1 takes the residual value, but z^1 may be 0] General formula (
2) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the above formula, R^2^1 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R^2^2 is -CH=CH_2 or -CH_2CH_2
X^2 (where X^2 represents a group that can be substituted by a nucleophilic group or a group that can be eliminated in the form of HX^2 with a base); L^2 is 1 to 6 carbon atoms an alkylene group having from 6 to 12 carbon atoms, an arylene group having from 6 to 12 carbon atoms, -C
A group represented by OZ^2-, and -COZ^2R^3
A group represented by ^5- (where R^3^5 is 1 to 6
an alkylene group having 6 to 1 carbon atoms, or 6 to 1
is an arylene group having 2 carbon atoms, and Z
^2 is a divalent group selected from (oxygen atom or NH); A^2 is a divalent group derived from an ethylenically unsaturated monomer copolymerizable with each monomer unit shown on the left side. and x^2, y^2 and z^2 represent molar percentages, and x
^2 is 50 to 99, y^2 is 1 to 50, z^2 takes the residual value, but z^2 may be 0] and an acrylamide-based compound are cross-linked and polymerized in the presence of water. A polyacrylamide-based aqueous gel electrophoresis medium. 2. The polyacrylamide-based aqueous gel is obtained by cross-linking and polymerizing the methacrylamide-based copolymer, an acrylamide-based compound, and further a cross-linking agent in the presence of water. medium for electrophoresis. 3. The electrophoresis medium according to claim 2, wherein the crosslinking agent is a trifunctional crosslinking agent. 4. The electrophoresis medium according to claim 1, further comprising a water-soluble polymer and agarose. 5. The electrophoresis medium according to claim 1, further comprising an anionic surfactant as a denaturing agent. 6. The electrophoresis medium according to claim 4, wherein the anionic surfactant is an alkyl sulfate. 7. The electrophoresis medium according to claim 6, wherein the anionic surfactant is sodium dodecyl sulfate. 8. The electrophoresis medium according to any one of claims 1 to 7, which is attached to a support made of plastic material.