JPS63233361A - Medium material for electrophoresis - Google Patents

Abstract

PURPOSE:To enhance bonding strength, by successively laminating a support layer, an adhesive layer containing a polymer having a constitutional repeating unit represented by a specific formula and an electrophoretic medium layer containing a polyacrylamide type aqueous gel prepared by the crosslinking polymerization of an acrylamide type compound and a crosslinking agent in the presence of water. CONSTITUTION:An adhesive layer containing a polymer having a constitutional repeating unit represented by formula (wherein r<11> is a hydrogen atom or a 1-6C alkyl group Q<1> is divalent group such as a 6-11C arylene group, L<1> is a 3-15C divalent group containing at least one group such as -COO-, R<12> is -CH=CH2m, A<1> is a divalent group derived from an ethylenic unsaturated monomer copolymerizable with the monomer shown on the left side of R<12>, x<1>, y<1> and z<1> shown mol. percentage and x<1> is 0-99, y<1> is 1-99 and z<1> is 1-99) is formed on a support layer such as a plastic sheet. An aqueous gel medium layer wherein an acrylamide type compound and a crosslinking agent are subjected to crosslinking polymerization in water is formed thereon to obtain an electrophoretic medium material having high bonding strength.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a medium material for electrophoresis, and more specifically, a medium material suitable for use in electrophoretic analysis of biopolymer components such as proteins. This is related to electrophoresis 1 model.

[Prior Art] A typical mode of electrophoretic analysis is to coat or cast a film-forming material such as agar, cellulose, cellulose acetate, starch, silica gel, or polyacrylamide on a support such as a glass plate or transparent plastic. The manufactured electrophoretic membrane is impregnated with a buffer solution, the substance to be analyzed is attached onto it, a voltage is applied to both ends of the support, it is developed (moved) on or inside the support, and then dyed. One example of this method is to measure the optical density of the stained sample to perform quantitative analysis of each component of the substance.

For details on electrophoretic analysis and electrophoretic membranes, please refer to the Electrophoretic Experimental Methods "Electrophoretic Experimental Methods (Revised 5th Edition)" (
Bunkodo, published in 1975), "Latest Electrophoresis Methods" edited by Kapital and Mizuben (Yokoyo Shoten, published in 1973), etc.

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

On the other hand, as a membrane or sheet for electrophoresis, from -i<,
Paper was previously used, but recently agarose membranes and polyacrylamide gel membranes have been used due to their properties, and polyacrylamide gel membranes are currently the most commonly used because they have a molecular sieving effect. There is. Polyacrylamide gel membranes are obtained by polymerizing and crosslinking monomers such as acrylamide with a difunctional crosslinking agent such as N,N''-methylenebisacrylamide in the presence of a polymerization catalyst in the absence of oxygen. It is being

In addition, an anionic surfactant is added as a denaturing agent when producing this polyacrylamide gel membrane, but in the case of gel membranes for protein analysis, the amount of denaturing agent added is small.
The modifier can be impregnated into the gel film by a method such as applying an aqueous modifier solution to the wet gel film or immersing the gel film in the aqueous modifier solution.

As mentioned above, since the polymerization reaction for forming polyacrylamide is radical crosslinking polymerization, crosslinking polymerization is inhibited by the influence of oxygen. Therefore, the gel film must be created in a state where oxygen is blocked. For this reason, at present, polyacrylamide gel membranes are generally produced by injecting a gel-forming solution into a cell formed by two glass plates (a fixed space of 0.3 mm to 1 mm), blocking oxygen, and then crosslinking polymerization. It is formed by allowing the mixture to gel. However, the process of forming a gel film between two glass plates has drawbacks such as the glass being easily broken and being heavy, as well as being inconvenient to carry and difficult to handle, making mass production extremely difficult. Accompany.

In order to solve these difficulties, it is desired to develop a polyacrylamide gel membrane provided on a light plastic support instead of a glass support. However, in this development, as will be described later, it is necessary to improve the adhesion between the gel film and a plastic support that is easy to handle, such as a polyethylene terephthalate (PET) sheet.

That is, in the past, horizontal or vertical slab electrophoresis was performed using a polyacrylamide gel membrane sandwiched between glass plates for a certain period of time under predetermined conditions, and then the gel membrane was stained (for example, Denso 3R staining, Coomassie Brilliant Blue G).
-250 staining, silver staining, etc.) and analysis of biological components, but due to poor adhesion between the glass plate and the gel film, the gel film peels off from the glass plate during this staining process. However, it has the disadvantage of extremely poor handling. Therefore, this work requires highly skilled techniques.

Provides an adhesive layer coating solution for increasing the adhesiveness (increasing adhesive force) between a gel film and a support in a wet state, and further,
An invention aimed at providing a gel film that is difficult to peel off from a support even in the dyeing step after electrophoresis or the drying step after dyeing treatment is described in JP-A-59-212752,
However, the gel medium according to this invention cannot be said to have sufficiently satisfactory adhesion and non-peelability.

[Object of the Invention] An object of the present invention is to provide a coating liquid having sufficient adhesive strength, and further to provide a gel film having sufficient resistance to peeling during the dyeing and drying process.

[Structure of the invention] The present invention is.

[I] Support layer; [II] Adhesive layer containing a polymer having a constituent repeating unit represented by a specific general formula; and [m] An acrylamide-based compound and a crosslinking agent are crosslinked and polymerized in the presence of water. The electrophoretic medium material has a three-layer structure in which the electrophoretic medium layer includes a polyacrylamide-based aqueous gel and is sequentially laminated.

As described above, the electrophoretic medium material of the present invention includes a three-layer structure in which the support layer and the electrophoretic medium layer are bonded together by a specific adhesive layer, and the electrophoretic medium layer (
The three-layer structure is difficult to separate even during various operations in the dyeing process of the gel membrane, which is very advantageous in terms of work.

Furthermore, the electrophoresis medium material of the present invention can also be produced by a method in which an adhesive layer is formed on a horizontal support and then an electrophoresis medium layer is formed thereon. This will greatly contribute to the mass production of electrophoretic media materials.

Next, the present invention will be explained in detail.

As a support for the electrophoresis medium material of the present invention, a glass plate, paper, or various plastic sheets can be used, but in order to take advantage of the advantages of the present invention, a plastic sheet is used as the support. Preferably, the plastic sheet may be made of any plastic, but
In particular, it is preferable to use a sheet made of polyethylene terephthalate.

In the present invention, an adhesive layer is formed on the support.

The adhesive layer is a layer containing a polymer having a structural repeating unit represented by any of the following general formulas (1) to (3).

[In general formula (1), R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; ■ Ql is -coo-1-CON-1 or arylene having 6 to 10 carbon atoms; A divalent group containing at least one of the following groups and having from 3 to 15g1 carbon atoms, or R1+ 10-1-N-1-CO-1-SO-1-SO□-1-
SO, -, -3O2N-1-NCON-1 or -N
It is a divalent group containing at least one bond of COO- and having 1 to 12 carbon atoms (however, 1li11 has the same meaning as above); ) i12 is -CH=CH2 or -CH , CH2X1 (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): AI can be copolymerized with one unit of each monomer shown on the left side. It is a 92-valent group derived from an ethylenically unsaturated heptamer; Xl, yl and zl represent molar percentages, and xl is O
~99, yl takes a value of 1 to 99, and zl takes a value of 1 to 99] In the above general formula (1), examples of the Rth group include a methyl group, an ethyl group, and a butyl group.

Contains n-hexyl group.

Examples of Ql include the following groups:

Examples of uff I -Coo-1-CONH-, -CON-1Ll include 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.

-CH2COOCR2- -CH, C00CH, CH2- -CH2CH, C00CH2- fCH, cheese C00CH, CH2- -fCH2 loaf,
. COOCH2CH2--CH,NHCOCH,--CH2NHCOCH2CH2- (CH2de3N HCOCH2C)12-fCH2
Chi S NHCOCH2CH, -i CH2 catty. N H
COCH2CH2--(:, H20CH2- -CH2CH20CH2CH2CH2--C0CH, C
H, --CH2COCH2CH2--ch2--ch, 5Och2ch2--s 02 CH2CH2 -S O2ch2ch2S O2ch2ch2 -h L -3-0 -C) 12ch2ch2- -S OS CH2COOCH2CHT- -3O3ch
2CH2C00CH,CH,--502NHCH,C0
0CH2CH2--SO□ N HCH2CH2COO
CH2CH2--N HCON HCH2CH2- -CH2N HCON HCH2CH2--N HCO
As mentioned above, OCH2CH2- CH2N HCOOCH2CH2- 11i12 is either -CH=CH2 or -CH, CH, or XI, and examples of -XI include the following groups.

Halogen atoms (chlorine, bromine, etc.); Hydroxyl group; Alkylsulfonyloxy (e.g., methylsulfonyloxy H3C-S-0-, ethylsulfonyloxy, propylsulfonyloxy, etc.); Arylsulfonyloxy (
Examples, phenylsulfosulfonyloxy, etc.): alkylsulfonyloxy (eg, acetoxy, propionyloxy, trifluoromethylcarbonyloxy, dichloromethylcarbonyloxy, etc.) Therefore, examples of R12 include the following groups.

-CH=CH2, -CH2CH2C4Q, -CH2C
H2Br, -CH2CH20S CH, -CH2CH2
0H1 = CH2CH200CCHz, CH2CH200CCF s.

-CH2CH200CCHCl2.

Examples of the divalent group represented by A1 include groups derived from ethylenically unsaturated monomers as shown below.

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, l-vinyl-2-methylimidazole, monoethylenically unsaturated esters of aliphatic acids (e.g. vinyl acetate, allyl acetate), ethylenically unsaturated monocarboxylic Acids 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, Cyanoethyl 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-methacryloyloxy Ethylammonium p-)luenesulfonate, N,N-diethyl-N-methyl-N-methacryloyloxyethylammonium P-toluenesulfonate, dimethyl itaconate, maleic acid monobenzyl ester), ethylenically unsaturated monocarboxylic acid or amides of dicarboxylic acids (e.g. N,N-dimethylacrylamide, N-methylolacrylamide, N,-[(dimethylamino)propylcoacrylamide, N,N,N-
) Limethyl-N-(acryloylpropyl)ammonium, p-toluenesulfonate, 2-acrylamide-
Sodium 2-methylpropanesulfonate, acryloylmorpholine, methacrylamide, N,N-dimethyl-
No-acryloylpropanediamine propionate betaine, N,N-dimethyl-No-methacryloylpropanediamine acetate betaine.

When the polymer of the present invention is used as a crosslinked latex, Al can be used in addition to the groups derived from the ethylenically unsaturated monomers mentioned above.

A monomer having at least two or more copolymerizable ethylenically undisturbed groups (eg, divinylbenzene).

Methylene bisacrylamide, ethylene glycol diacrylate, trimethylene glycol diacrylate,
Groups derived from ethylene glycol dimethacrylate, trimethylene glycol dimethacrylate, neopentyl glycol dimethacrylate, etc.) can be used.

[In general formula (2), R21 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms: R22 is -CH=CH2 or -CH20M2X2 (however, x2 may 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 6 carbon atoms from 1 (
e.g., methylene, ethylene, imbutylene), 6 to 12
Examples of arylene MC having 1 to 6 carbon atoms: phenylene, tolylene, naphthalene), a group represented by -COZ2-, and a group represented by -COZ2R23- (where R23 is an alkylene group having 1 to 6 carbon atoms) , or an arylene group having 6 to 12 carbon atoms, where Z2 is an oxygen atom or NH); is a divalent group derived from an ethylenically unsaturated heptamer copolymerizable with takes a value of 1 to 99] In addition, R21,1 (22, and A
Examples of 2 include R11 and R1 of the general formula (1), respectively.
The same examples as 2 and A1 are included.

[In general formula (3), 1 (31 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; L3 is a divalent linkage 7! having 1 to 20 carbon atoms)
i (more preferably a divalent group having 1 to 2 carbon atoms containing at least one of -CONH- or -Co- bond)
is; z3 represents mole percentage.

X-co is 0-99, y-co is 1-99
, Z co takes a value of 1 to 99; m is O or 1] R11 and A! of the general formula (1) are respectively substituted for 1li31 and A3 of the general formula (3) written in L! Contains the same things as the example.

L3 in general formula (3) includes the following groups.

-COCH, - -C0CH, CH20COCH2CH2- -CONHC
H2- CON HCH2CHt - -CONHCH,CH,CB, - -CON HCH2CHt CH2CH2CH2--C
ONHCH2C0NHCH, - -C0NHCH, C0NHCH2CONHCH2 - -C
0NHCH2NHCOCH,CH,CH,S 0H2c
ut --C0NHCH, 000CI 2CI, --
x3 in general formula (3) includes the following groups.

-COOCR2CN -COOCHt COOC2Hs -〇 〇 〇 〇 Hz CON H2-COON =
CHCH3 -COO'N = CCCH3) 2-COOC=
CHCOCH3 -COOCH2CH2B r -COOCH, CH, CN -COOCH2CHt N -CHs 0 sentences H3 Below margin To be used for the synthesis of a polymer having a structure J&Kakigaeshi unit represented by any of the above general formulas (1) to (3) Regarding the synthesis method of an ethylenically unsaturated monomer having a typical vinylsulfonyl group or a functional group which is a precursor thereof.

A detailed description is given in JP-A-59-212752.

Preferred polymers for forming the gel medium of the present invention include polymers having the following constituent repeating units.

Here, x, y, and z are molar percentages, and RlRo represents the following substituent.

A-1x=80.7=8, z=12R=000C
H2CH20COCHt (dot 5o2(d(=
CH2R' = C0NHG(CHs) 2 GHz
C0CR3A-2x=80, y=8. z=12 R= C0NHCHz NHCOC)It CH2'J
Ot CH= CH2R' = C0NIC(OH3
) 20H2C0CH3A-3x=80.7='8.
z=12 R= C0NHCH2NHGOCH2CH2SO2CI
2 CH2CIR' = C0NHC(CH3)
2 CH2C0CH5R' = C0N(CHz
) 2A-Ei x=70, y=5. z
=25R'= CoN(CHri) 2 A-? x=50.7=5. z=45 By the way, JP-A-59-212752 discloses a polymer using an acrylamide component instead of the methacrylamide component of the present invention.

Here, x, y, and z are mole percentages, and R5R° represents the following substituent.

B-1x=80.7=8, z=12R=Coo
CH, C1,000CH2CH25O□C)I=C)I
2R' =cON)IC(C)1ri) 2 G'Ht
C0CH B-2x=80.7=8, z=12 R=C0NHCH2N)lcOcH2CH2S02 C
I=CH2R'=C0NIC(CHz) 2
CH2C0CH5B-3x=80.7=8. z=12 R= C0NHCH, NHCOCH, CH, So□0H
20H, CIR' = C0NIC (CH) 2
CHt COCHsR' = C0N1[H2
0H R' = C0N(CH3) 2B-6x=7
0, y=5. z=25R'=C0N(
CHs) 2B-7x=50. y=5, z=
45 Next, synthesis examples of A-1 and A-3, which are particularly preferred copolymers among the above copolymers, will be shown.

[Synthesis Example 1] Copoly-2-[3-(vinylsulfonyl)propionyloxy]ethyl acrylate-methacrylamide-N-
Synthesis of (1, t-dimethyl-3-oxbutyl)acrylamide (A-1) 80 ml of N,N-dimethylformamide was added to the reaction vessel.1.2-[3-(2-chloroethylsulfonyl)propionyloxy]ethyl acrylate 14
．． 5g, methacrylamide 41.3g, N-(1,1
-dimethyl-3-oxbutyl)acrylamide12.
After adding 3g and degassing with nitrogen gas.

Heating to 60°C, 2,2°-azobis(2,4-dimethylvaleronitrile) [CAS Register
ryNo, 4419-11-8] 0, 40 g
was added, and heating and stirring was continued for 2 hours. After that, 2,
2°-azobis(2,4-dimethylvaleronitrile)0
．． After adding 20g of sodium carbonate and continuing to heat and stir for 2 hours, the mixture was cooled to 5°C and 12g of sodium carbonate and 4.0g of triethylamine were added.
9 g was added and stirred for 1 hour. After further stirring at room temperature for 2 hours, the reaction sample was placed in a cellulose tube and dialyzed for 2 days, and 64 g of white polymer (corresponding to A-1 above) was obtained by freeze-drying (yield 90%). The vinylsulfonyl group content of the copolymer is 0.8X10-
[Synthesis Example 2] Copoly-N-([3-(vinylsulfonyl)propanamidocomethyl)acrylamide-methacrylamide-N-(1,l-dimethyl-3-oxobutyl)acrylamide ( Synthesis of A-3) In a 500m reaction vessel, 10.3 g of N-[3-(2-chloroethylsulfonyl)propanamidomethyl], 31.0 g of methacrylamide, and N-(1,l-dimethyl-
Add 9.3 g of 3-oxobutyl)acrylamide and 160 ml of 50% methanol aqueous solution, and heat to 60°C while stirring.
0.2 g of 2,2゛-azobis(2,4-dimethylvaleronitrile) was added, and 0.2 g of the same was added after 30 minutes, and heating and stirring was continued for 2 hours. It was dialyzed in a cellulose tube for 2 days, and 46 g of a white polymer (corresponding to A-3 above) was obtained by freeze-drying (yield 90%). The vinyl sulfonyl group content of this copolymer was 0.7XIO-' equivalent/ It was g.

Next, the gel medium layer of the present invention will be described.

In the present invention, the acrylamide compound and the crosslinking agent are
It is dissolved or dispersed in water as an aqueous solution or an aqueous dispersion, and both are crosslinked and polymerized in water to form a crosslinked and polymerized aqueous gel medium layer.

In this specification, unless otherwise specified, "in water" simply means "dissolved" (in water) and "dispersed" (in water).
The term "dissolution" includes both an aqueous solution and an aqueous dispersion, and also includes a mixture of an organic solvent and wood, which may be added as a solvent or dispersion medium if desired.

Acrylamide compounds that can be used in the present invention include acrylamide, N-methylacrylamide,
Examples include acrylamide compounds such as N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide, and diacetone acrylamide, 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.

As a crosslinking agent, r Electrophoresis
J 1981°2, 213-228, etc. (one type or a combination of two or more types) can be used. Specific examples of crosslinking agents include N,N'-methylenebisacrylamide (BIS); N,N'-propylenebisacrylamide (PBA), di(acrylamide dimethyl) ether (DAE); l,2-diacrylamide ethylene glycol. (DEC).

Ethyleneureabisacrylamide (EUB).

Ethylene diacrylate (EDA), N,N'-diallyltardiamide (N,N'-diallyltar)
tardiamide: D A TD);
N,N'-bisacrylylcystamine (N,N'
-bigacrylylcystamine, B A
Bifunctional compounds such as C) can be mentioned.

The amount of crosslinking agent is approximately 2w based on the total weight of monomer and crosslinking agent.
t% to about 30 wt%, preferably about 3 wt% to about 1
It can be used in a range of 0 wt%.

The gel concentration is S, Hjerten: r
Arch.

Biachem, Biopbys-J 1 (
5upp1. ), 147 (1962), where the amount of monomer and crosslinker is approximately 3% relative to the volume of the gel medium consisting of monomer, crosslinker and wood.
It is preferably used in a range of w/v% to about 30 w/v%.

The electrophoresis medium material of the present invention is mainly used for analyzing proteins or complex proteins (e.g. riboprotein, glycoprotein, etc.), and the electrophoresis medium layer (hereinafter also referred to as gel medium) contains: An anionic surfactant can be included as a denaturing agent.0 If the analysis sample is a protein or complex protein (e.g. riboprotein, glycoprotein, etc.), it is preferable to include an anionic surfactant, or Often required.

Examples of the anionic surfactant include alkyl sulfates, and in particular, alkyl sulfates having a long chain alkyl group having 10 or more carbon atoms are preferably used. Cations that form salts include sodium ions, potassium ions,
Alkali metal ions such as lithium ions are common, and among these, sodium ions are cheap and easy to use.Among alkyl sulfates, dodecyl sulfates (sodium salts, potassium salts, lithium salts, etc.) are preferred, and among these, dodecyl sulfates are preferred. The inclusion of SO3, most preferably sodium sulfate (SOS), in the gel media layer of the present invention allows for the separation of proteins or complex proteins and the determination of their molecular weight.

The content of the anionic surfactant as a denaturing agent is about 0.05 w/v% to about 2 Ow/v%, based on the gel forming solution.
Preferably from about 0,1 w/V% to about 1.5 w/v
% range. It goes without saying that a gel medium layer that does not contain an anionic surfactant as a modifier can also be used.

Note that other surfactants may be added to the gel medium layer of the present invention, and examples of such surfactants are described in Japanese Patent Application No. 145480/1983.

In the present invention, the polyacrylamide-based aqueous gel medium layer can contain a water-soluble polymer.

As the water-soluble polymer, addition polymerization type or condensation polymerization type water-soluble polymer can be used.

Specific examples of addition polymers include nonionic water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide. Specific examples of condensation polymers include nonionic water-soluble polyalkylene glycols such as polyethylene glycol and polypropylene glycol. The molecular weight of the water-soluble polymer is preferably in the range of about 10,000 to about 1 million. Among these water-soluble polymers, polyethylene glycol and polyacrylamide are particularly preferred.

The water-soluble polymer is used in a range of about 0.1 wt% to about 100 wt%, preferably in a range of about 2 wt% to about 50 wt%, based on the total weight of monomer and crosslinker.

In the present invention, by adding a water-soluble polymer, the gel medium layer has plasticity and does not break during cutting, and the gel medium layer also has plasticity when drying, reducing brittleness. It has the advantage of being improved and less likely to break. The viscosity of the gel medium can also be controlled by selecting the molecular weight and amount of the water-soluble polymer added.

In the present invention, the gel medium layer can contain agarose. The agarose is not particularly limited as long as it is a known agarose, and any of low electroosmotic, medium electroosmotic, and high electroosmotic agaroses can be used. Examples of agarose that can be used include those disclosed in Japanese Patent Application Laid-open Nos. 57-5730, 110946-1982, and Japanese Patent Publication No. 502098-1980.

The amount of agarose added is about 0.2 w/v% to about 2 w/v% based on the volume of the gel composition solution containing the monomer and the crosslinking agent.
, preferably from about 0.3 w/v% to about 1.2 w/v%
It is used at a ratio of v%.

In Undeveloped 11, 7galose is added to the gel medium layer, so by changing the temperature of the gel-forming solution, it is necessary to control the solution viscosity to an appropriate level, and the fluidity can be stopped. It also has the advantage of being easier to mold when molding the gel medium layer.

The gel medium layer of the present invention can contain a pH buffering agent. The buffer can be appropriately selected from known buffers capable of buffering to a pH value within the range of pH 2.5 to 10.0, depending on the sample to be electrophoretically analyzed.

As a buffering agent that can be used, Nippon Kagaku complete edition "Chemistry Handbook Basic Edition" (Tokyo, Maruhata @ Published in 1966) 1312-1
Page 320: “Latest Electrophoresis Methods” edited by Furuki and Mizuben (Tokyo, Tokoyo Shoten, published in 1973, pages 320-322;
r Data far Bioche+5ical
Re5earch J (R.

M. C. Dawson et al., ed., 2nd edition, 0
xford at the C1arendon Pre
ss, published in 1969) pages 4711i-508; [formerly ochemistr7 J 5.467
(19611i).

rAnalytical Biochemistry
J 104.300-310 (1980) and the like.

Specific examples of buffers include buffers containing barbital,
Tris(hydroxymethyl)aminomethane (Tris)
buffers containing phosphate, buffers containing borate, buffers containing acetic acid or acetate, buffers containing citric acid or citrate, buffers containing lactic acid or lactate, glycine A buffer containing N,N-bis(2-hydroxyethyl)glycine (Bicine), N-2-hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid (HEPPSO) or a salt thereof, N
-2-Hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS) or its salt, N-[
tris(hydroxymethyl)]-]3-7minopropanesulfonic acid TA PS) or a salt thereof, and an acid, alkali,
Or salt etc. can be mentioned. Specific examples of preferred buffers include potassium dihydrogen phosphate-disodium wood phosphate, Tris-sodium borate, and Tris-sodium borate.
Sodium borate-EDTA-2Naa1. Tris-
Citric acid, parbital sodium-sodium acetate,
Parbital sodium-hydrochloric acid, parbital sodium barbital, acetic acid-sodium acetate, lactic acid-sodium lactate, citric acid-disodium hydrogen phosphate, Bic
ine, HEPPSO, HEPPSO sodium salt, E
PPS, EPPS sodium salt, TAPS.

Examples include TAPS sodium salt.

In the present invention, the gel medium layer is formed in an aqueous solution in which a monomer typified by acrylamide, a difunctional allyl compound or acrylic compound (crosslinking agent), a water-soluble polymer, and agarose are substantially uniformly dissolved. It is obtained by radical crosslinking polymerization of a monomer and a crosslinking agent, and a water-soluble polymer and 7 galose are substantially dispersed in a three-dimensional crosslinked polymer formed from a monomer and a crosslinking agent. It is presumed that the latter two polymer buttons have a structure in which they are intertwined with a three-dimensional crosslinked polymer, and this structure is the characteristic gel medium layer.

The above-mentioned radical crosslinking polymerization reaction can be caused in the absence of a molecular acid by a known method such as the presence of a peroxide and/or ultraviolet irradiation. This reaction can also be accelerated by heating and UV radiation.

As a catalyst for radical crosslinking polymerization, rElectr.

phoresisJ 1981.2.213-219.
1981.2.220-228; Seimizu and Mizuben eds., "Latest Electrophoresis Methods J" (published in 1973), etc., known low-temperature radical polymerization initiators can be appropriately selected and used.

Specific examples of preferred radical polymerization initiators include β-dimethylamine propionitrile (DMAPN)-ammonium peroxonisulfate mixture, N, N, N', N'
-Tetramethylethylenediamine (TEMED)-peroxoni'i & ammonium acid mixture, TEMED-riboflavin mixture, combination of TEMED-riboflavin-hydrogen peroxide mixture and ultraviolet irradiation. The content of the radical polymerization initiator ranges from about 0.3% to about 5% by weight, and preferably from about 0.5% to about 3% by weight, based on the total weight of monomer and crosslinking agent. be.

The radical polymerization initiation means applied to the method for producing an electrophoresis medium of the present invention includes r Electr.

phoresisJ 2(4) 213-219(
1981) Same magazine” (0220-228 (1981)
In addition to the combination of peroxide and photosensitizer used in the low-temperature radical polymerization initiator composition described in JP-A-59-126236, edited by Aomizu and Mizuben, "Latest Electrophoresis Method J" (published in 1973), etc. Combinations of excitation lights can also be used.

Specific examples of peroxides include ammonium belloxonisulfate, alkali metal salts of belloxonisulfate (examples of alkali metals, such as sodium and potassium), hydrogen peroxide, and the like.

Specific examples of photosensitizers include riboflavin. Examples of the radical polymerization initiator composition include a lipoflavin-beroxonisulfate mixture, a riboflavin-hydrogen peroxide mixture, and the like. The amount of the radical polymerization agent composition added is about 0.3 w% to the total weight of the monomer and crosslinking agent.
It ranges from about 5.0 w%, preferably from about 0.5 to about 3.0 w%.

The excitation light has a wavelength of approximately 200n, which is effective for excitation of the photosensitizer.
Any light in the range from m to about 900 nm (range from near ultraviolet rays to rIf visible light) can be used without limitation. The intensity and irradiation time of the excitation light may be appropriately selected so that the gel-forming liquid gels substantially uniformly in the thickness direction of the layer.
Specific examples of the excitation light source include a high-pressure mercury lamp, an ultra-high-pressure mercury lamp, a low-pressure mercury lamp, a halogen lamp, a metal halide lamp, a tungsten lamp, a deuterium lamp, and a xenon discharge lamp.

In the present invention, the gel medium layer is formed by applying a gel forming liquid by a known method onto the above-mentioned adhesive layer provided on an electrically insulating support having a smooth upper surface. It can be formed into a gel layer by cross-linking polymerization.

The support has been described above, but more specifically, a glass plate, a wood-philic polymer, or a polymer whose surface has been made hydrophilic by known surface treatment (e.g., polyethylene terephthalate, polycarbonate of bisphenol A, polyvinyl chloride, Examples include molded products such as films, plates, and sheets of vinylidene chloride/vinyl chloride copolymers, polymethyl methacrylate, polyethylene, polypropylene, cellulose acetates, cellulose acetate flobionate, etc.). As a treatment for making the surface of these polymer moldings hydrophilic, known methods such as ultraviolet irradiation, glow discharge treatment, corona discharge treatment, flame treatment, electron beam irradiation, chemical etching, electrolytic etching, etc. can be applied. can.

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. The cover film, sheet, or plate used for this purpose may be made of the same material as the support. The thickness of this coating material is 300 gm or less, and the practically preferred range is about 41 Lm to about 200 JLm.
, a particularly preferred range is from about 4 gm to about 100 pm.

The coating material is thick (e.g., about 70jLm to about 300pm)
), a method in which a gel-forming liquid is applied onto the coating material, a gel medium layer is formed by cross-linking polymerization, and then a support provided with the adhesive layer of the present invention is laminated to form a three-layer structure. You can also use

The gel medium layer can contain an antioxidant if necessary. As the antioxidant, various compounds known to be able to be incorporated into gel electrophoresis media can be used. Specific examples of the antioxidant include dithiothreitol and 2-mercaptoethanol.

As other additives, polyol compounds such as glycerin and ethylene glycol can also be included in the gel medium layer for the purpose of wetting agents and improving the electrophoretic image. What is the content of polyol compounds?

Approximately 0.0% relative to the volume of gel medium. lW/V% to about 40
w/v%, preferably from about 0,5 W/V%
It is selected from a range of about 40 W/V%. Among the polyol compounds, glycerin is particularly preferred. By blending the polyol, it is possible to prevent the gel medium layer from drying out due to extreme water evaporation during storage, and also prevents brittleness caused by extreme drying and prevents cracking. There is an advantage that the physical properties of the gel medium layer, such as prevention, are improved.

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

[Example 1 and Comparative Example 1] The above-mentioned material was placed on a polyethylene terephthalate (PET) sheet (support) whose surface had been made wood-philic by ultraviolet irradiation treatment to a thickness of about 0.5 JLm (solid content). Copolymers A-1, A-3, A-8° B-1% 5 g each of B-3 and B-6 were applied and dried at about 110°C.
A support with an adhesive layer was produced. Sample each sample! , 2.3.4.5, and 6.

First, regarding the PET sheet with an adhesive layer (support), the adhesiveness between the support and the adhesive layer was evaluated by a cross-cut method. As a result, sample number l.

For samples Nos. 2 and 3 (samples according to the invention), 0-5% peeling was observed between the sheet and the adhesive layer, whereas for sample numbers 4, 5 and 6 (comparative samples), 15%-5% peeling was observed between the sheet and the adhesive layer.
Approximately 20% peeling was observed.

On top of each adhesive layer provided on the support, 9.5 g of acrylamide, 500 mg of BIS, 3.58 g of disodium hydrogen phosphate, 0.33 g of sodium dihydrogen phosphate, and SOS. 1.3 mJ of ammonium persulfate (5% by weight) as a polymerization initiator was added to 100 ml of a solution containing 0.10 g of ammonium persulfate (5% by weight). TEMED33g
1 was added and molded to a thickness of 0.5 mm to obtain a polyacrylamide gel film.

The obtained gel film was pressed with a finger to evaluate the adhesion between the gel film and the support. As a result, when sample numbers 1, 2, and 3 (samples according to the present invention) were used as an adhesive layer, the adhesive properties were excellent, but the adhesive properties were poor for sample numbers 4, 5, and 6 (comparative samples). .

[Example 2 and Comparative Example 2] Using the same polymer as in Example 1 and Comparative Example 1, a PET sheet (support) with an adhesive layer was manufactured.

On top of this adhesive layer, 9.5 g of acrylamide, BIS50
0mg, Agarose 1600 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 0.3
g, polyacrylamide 2.5 g, disodium hydrogen phosphate 3.58 g, sodium dihydrogen phosphate 0.33 g, and SDS O, lOg as a polymerization initiator. Ammonium persulfate (5% by weight) 1.3mJL, TEMED33ILJ
The product to which L was added was molded to a thickness of 0.5 mm to obtain a polyacrylamide gel film.

Using this gel membrane, standard proteins were subjected to electrophoresis.Next, the gel membrane was coated with 0.1% Coamseap
Asie Blue) R-250 for dyeing, and the state of adhesion between the support and the gel film in this dyeing process was observed.

The gel film formed on the adhesive layer of sample numbers 4 to 6 (comparative samples) was adhered to the support at the beginning of the dyeing process, but part of it peeled off from the support at the end of the process. was.

On the other hand, the gel films formed on the adhesive layers of sample numbers 1 to 3 (samples according to the present invention) were completely adhered to the support throughout the dyeing process. Furthermore, there were no problems with any of the electrophoretic properties.

[Example 3 and Comparative Example 3] An adhesive layer was prepared in the same manner as in Example 2 and Comparative Example 2! PET
A polyacrylamide gel film was formed on the adhesive layer of the sheet. After cutting this gel film along with the support, we observed the cut section of the gel film, and found that some of the gel films formed on the adhesive layer of sample numbers 4 to 6 (comparative samples) were peeled off. On the other hand, it was found that all sample numbers 1 to 3 (samples according to the present invention) had good adhesion at the cutting edge, and could be cut together with the support.

Claims (1)

[Claims] 1. An electrophoretic medium material comprising a three-layer structure in which the following layers are sequentially laminated: [I] Support layer: [II] Any one of general formulas (1) to (3) Adhesive layer containing a polymer having a constituent repeating unit represented by: ￥General formula (1)￥ ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In general formula (1), R^1^1 is a hydrogen atom or 1 Q^1 is an alkyl group having 6 carbon atoms from , L^1 is a divalent group containing at least one of the bonds -COO- or ▲which has a mathematical formula, chemical formula, table, etc.▼, and has 3 to 15 carbon atoms, or
-O-, ▲Mathematical 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 (however, R^1^1 has the same meaning as above); R ^1^2 is -CH=CH_2 or -CH_2CH_2
Any of is a divalent group derived from an ethylenically unsaturated monomer copolymerizable with each monomer unit; x^1, y^1 and z^1 represent molar percentages, x
^1 is 0-99, y^1 is 1-99, z^1 is 1-99
] ￥ General formula (2) ￥ ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ [In general formula (2), R^2^1 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. is; 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 removed by a base in the form of HX^2); 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^2
A group represented by ^3- (where R^2^3 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 an 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. is; x^2, y^2 and z^2 represent molar percentages, and x
^2 is 0-99, y^2 is 1-99, z^2 is 1-99
] ￥ General formula (3) ￥ ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ [In general formula (3), R^3^1 is a hydrogen atom or an alkyl having 1 to 6 carbon atoms is a group; L^3 is a divalent linking group having 1 to 20 carbon atoms; X^3 is an active ester group; A^3 is copolymerized with each monomer unit shown to its left is a divalent group derived from possible ethylenically unsaturated monomers; x^3, y^3 and z^3 represent molar percentages, x
^3 is 0-99, y^3 is 1-99, z^3 is 1-99
m is 0 or 1] [III] An electrophoretic medium layer comprising an aqueous polyacrylamide gel formed by crosslinking polymerization of an acrylamide compound and a crosslinking agent in the presence of water. 2. The electrophoresis medium material according to claim 1, wherein the electrophoresis medium layer further contains a water-soluble polymer and agarose. 3. The electrophoretic medium material according to claim 1 or 2, wherein the electrophoretic medium layer further contains an anionic surfactant as a denaturing agent. 4. The electrophoretic medium material according to claim 3, wherein the anionic surfactant is an alkyl sulfate. 5. The electrophoretic medium material according to claim 3, wherein the anionic surfactant is sodium dodecyl sulfate. 6. The electrophoretic medium material according to claim 1 or 2, wherein the support layer is made of a plastic sheet. 7. The electrophoretic medium material according to claim 3, wherein the plastic sheet is made of polyethylene terephthalate. 8. The electrophoretic medium material according to claim 1, wherein the adhesive layer contains a polymer having a structural repeating unit of general formula (1).