JPS63228053A - Medium for electrophoresis - Google Patents

Abstract

PURPOSE:To provide a film which has a molecular sieving effect and sufficient physical strength by using an aq. gel contg. a specific methacrylamide copolymer as a medium for electrophoresis. CONSTITUTION:The aq. gel contg. the methacrylicamide copolymer having the constitutional repeating units expressed by formulas I, II, III is used as the medium for electrophoresis. In formulas I, II and III, R<11>, R<21>, R<31> denote H an alkyl group of 1-6C; Q<1> denotes -COO-, -CON(R<11>)-, arylene group of 6-10C; L<1> contains at least either of -COO- and -CON(R<11>)- and divalent combination group of 3-15C, etc.; R<12>, R<22> denote -CH=CH2, -CH2CH2X<1>, (X<1> is a group substd. by a nucleophilic group); A<1>, A<2>, A<3> denote a divalent group derived from an ethylenic unsatd. monomer, etc.; L<2> denotes alkyl of 1-6C, arylene group of 6-12C; L<3> denotes a divalent combination group of 1-20C; X<3> denotes an ester group. Since the aq. gel contg. such methacrylic amide copolymer is used, the medium for electrophoresis having high performance is obtd.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an aqueous gel electrophoresis medium used for electrophoretic analysis of biopolymers such as proteins, and more specifically, to an aqueous gel electrophoresis medium that can be used even in the presence of oxygen. The present invention relates to a manufacturable methacrylamide-based aqueous gel electrophoresis medium.

[Ban'an #Shibu] Electrophoretic analysis is performed by applying or casting a film-forming material such as agar, cellulose, cellulose acetate, starch, silica gel, or polyacrylamide gel onto a support such as a glass plate or transparent plastic. Impregnate the electrophoresis membrane with a buffer solution, or soak these electrophoresis membranes in a buffer solution,
Next, the substance to be analyzed (sample) is deposited on the electrophoretic membrane, a voltage is applied to both ends of the support, it is developed (moved) on the surface or inside the support, and then stained, and this stained sample is By measuring the (transmission) optical density of
It performs quantitative analysis of each component of a 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 Seimizu and Mizuben) (Yokoyo Shoten, published in 1973), etc.

In recent years, a method for analyzing biological components using electrophoresis has been established, while biological protein analysis is frequently used in biochemical tests for disease diagnosis, and the use of electrophoretic analysis is becoming increasingly important. Increasingly, its uses are also expanding.

By the way, filter paper has been used as a membrane or sheet for electrophoresis for a long time, but recently agarose membranes and polyacrylamide gel membranes have been used from the viewpoint of performance. Gel membranes are currently the most commonly used. Polyacrylamide gel membranes are produced by polymerizing monomers such as acrylamide in the presence of water with a difunctional crosslinking agent such as N,N''-methylenebisacrylamide in the presence of a polymerization catalyst under oxygen-free conditions. It is obtained by crosslinking.

The polyacrylamide film has a certain thickness (e.g. 0.3 m).
m~km) spacer on a glass plate, and after pouring an aqueous solution (gel-forming solution or gel stock solution) containing acrylamide, a crosslinking agent, and a polymerization catalyst into this r-hole, A gel film is created by pressing and sealing from above to block oxygen, and then cross-linking and polymerizing it to form a gel.

The polyacrylamide gel membrane created in this way is
Can be subjected to horizontal or vertical slab electrophoresis. After performing electrophoresis under predetermined conditions for a certain period of time, the gel membrane is stained (for example, Denso 3R staining, Coomassie Brilliant Blue G-250 staining, silver staining, etc.) to analyze biological components.

As mentioned above, in the production of conventional polyacrylamide gel membranes, oxygen removal operations are required, making it difficult to mass-produce gel membranes.

A gel medium that can be formed even in the presence of oxygen was disclosed in Japanese Unexamined Patent Publication No. 5
This medium is introduced in Japanese Patent No. 9-171849, but it has disadvantages such as a long time required for gelation because it is affected by oxygen to some extent, and the physical strength of the medium is low.

C. Purpose of the Invention] The purpose of the present invention is to provide a membrane-like aqueous gel medium that can further shorten the time required for gelation and has sufficient physical strength as an electrophoresis medium having a molecular sieving effect. There is a particular thing.

[Structure of the Invention] The present invention resides in an electrophoretic medium comprising an aqueous gel containing a methacrylamide copolymer having a constituent repeating unit represented by a specific general formula that is cross-linked and polymerized in the presence of water.

[Detailed Description of the Invention] The electrophoresis medium made of an aqueous gel of the present invention is a gel containing an acrylamide copolymer containing a structural repeating unit represented by any of the following general formulas (1) to (3). A medium is used. Particularly preferred is a polymer represented by general formula (1).

[In general formula (1), R11 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms: R11 Ql is -COO-1-CON-1 or an arylene group having 6 to 10 carbon atoms ,ll 11! LL is a divalent group containing at least one -COO- or -CON- (7) bond and having 3 to 15 carbon atoms, or 1i11 10-1-N-1- CO-1-SO-1! A divalent group containing at least one of -5O2-1-SO and -1-SO□N-1 and having 1 to 12 carbon atoms (however, R11 has the same meaning as above): R' bar CH= CH, or -CH2CH2X'
(However, xl represents a group substituted by a nucleophilic group or a group that can be eliminated in the form of HXI by a base); A1 is copolymerizable with each of the seven units shown on the left side. is a divalent group derived from an ethylenically unsaturated heptamer; xl, y'q and zl represent molar percentages, xl is 5
0-99. yl takes a value from 1 to 50, and zl takes a residual value (z
In the above general formula (1), examples of H include a methyl group, an ethyl group, a butyl group, and an n-hexyl group.

Examples of Ql include the following groups:

CH.

(2) Examples of -COO-, -CONH-, -CON-, and Ll include the following groups. In addition.

The bonding direction of these divalent groups in general formula (I) can be in any direction as long as the bonding between Ql and 802 is possible.

-CH2COOCH, - -CH2COOCH2CH2- -CH2CH2COOCH2- -(CH2te, C00CH2CH, - -(-CH2masu, .C00CH2CH2-CH2N H
COCH2- -CH2N HCOCH2CH2- (CH2 sho, NHCOCH2CH2- +CH2 辷s N HCOCH2CH2-(-CH2U-1゜N HCOCH2CH2- -CH20CH2- -CH, CH20CH2CH2CH2-CH, CH.

-NCH2CH2-CH2NCH2CH2--COCH
2CH2- -3OCR, CH2- -CH25OCH2CH2- -SO□ CH, CH, - 302CH2CH2S O2CH2CH2- -5o2C
H,2CH2So2CH2CHCH2■ H l -3-0-CH2CH2CH2- -50, CH, C00CH, CH, - -3O, CH2CH2C00CH, CH2--5O, N
HCH2C00CH, CH2--3O2NHCH2CH
2COOCH2CH2-NHCONHCH, CH2- -CH2NHCONHCH2CH, - -NHCOOCH2CH, - CH2N HCOOCH2CH2- As mentioned above, R12 is -CH=CH, or -CH
20H2Xi, but in this example of Xi,
Includes groups such as:

Halogen atoms (chlorine, bromine, etc.): Hydroxyl group; Alkylsulfonyloxy (e.g., methylsulfonyloxy H3C-3-0,-, ethylsulfonyloxy, propylsulfonyloxy, etc.); Arylsulfonyloxy (e.g., phenylsulfosulfonyloxy, etc.) ); alkylcarbonyloxy (eg, acetoxy, propionyloxy, trifluoromethylcarbonyloxy, dichloromethylcarbonyloxy, etc.) Therefore, examples of RL2 include the following groups.

-CH=CH2, -CH2CH2C standing, -CH2CH2
B r, -CH2CH2-0-S -CH3, -CH2CH20Hl -CH2CH200CCH3, -CH2CH200CCF s, -CH2CH200CCHC 7. Examples of the divalent group represented by A1 include the following ethylenically unsaturated groups: Examples include groups derived from.

Ethylene, propylene, 1-butene, isobutyne, styrene, chloromethylstyrene, hydroxymethylstyrene, sodium vinylbenzenesulfonate, sodium vinylbenzylsulfonate, N,N,N-trimethyl-
N-vinylbenzylammonium chloride, N,N-dimethyl-N-benzyl-N-vinylbenzylammonium chloride, α-methylstyrene, vinyltoluene, 4
-Vinylpyridine? -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-) diethyl-N-methacryloyloxyethylammonium p-) luenesulfonate,
N,N-diethyl-N-methyl-N-methac, lyloyloxyethylammonium 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)propylcoacrylamide, N , N, N-)limethyl-N-(acryloylpropyl)ammonium P
−) Luensulfonate, ? -Sodium acrylamide 2-methylpropanesulfonate, acryloylmorpholine, methacrylamide, N,N-dimethyl=N'-
acryloylpropanediamine probionate betaine, N,N-dimethyl-N'-methacryloylpropanediamine acetate betaine).

In addition, when the polymer of the present invention is used as a crosslinked latex, in addition to the groups derived from the above-mentioned ethylenically unsaturated monomers, a monomer having at least two or more copolymerizable ethylenically unsaturated groups is used as AX. (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.

[In general formula (2), R21 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R22 is -CH=CH, or -CH2CH2X2 (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 1.
Arylene groups having 2 carbon atoms (e.g., phenylene, tolylene, naphthalene), groups represented by -coz2-, and groups represented by -COZ 2 R2 (wherein R23 has 1 to 6 carbon atoms) alkylene group with

or an arylene group having 6 to 12 carbon atoms, and Z2 is an oxygen atom or NH); is a divalent group derived from a copolymerizable ethylenically unsaturated monomer: x2, y2 and z2 represent molar percentages, x2 is 5
0 to 95.72 takes the residual value to 5 to 50.22 (z
2 may be O)]: R21, R22 and A2 in the above general formula (2).

Examples include R11 and R12 of the general formula (1), respectively.
, and the same examples as Al.

[In general formula (3), R31 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; L3 is a divalent linking group having 1 to 20 carbon atoms (
More preferably, it is a divalent group having 1 to 2 carbon atoms containing at least one of -CONH- or -C〇- bond; x3 is an active ester group; A3 is each of the groups shown on the left side It is a divalent group derived from an ethylenically unsaturated monomer copolymerizable with one monomer unit: x3, y3 and z3 represent mole percentages, and X co is 5
0 to 99, y3 takes the residual value from 1 to 50.2 (z
3 may be 0): m is O or l] R318 and A in the above general formula (3) each include the same as the examples of R11 and Al in the above general formula (1). It will be done.

L3 in general formula (3) includes the following groups. −
COCH2- -COCH2CH20COCH2CH2--CONHC
H, - -CON HCH2CH2- CON HCH2G Hz CH2- CON HCH2CH2CH2CH2CH2- -CON
HCH2CONHCH2- -GONHCH2CONHCH2CONHCH2-CO
NHCH2NHCOCH2CH2CH2S CH2CH
2-CONHCH2000CH2CH2- etc. Formula (3)
x3 includes the following groups.

Lee O-COOC
R20N-COOCH2C00C,H.

-COOCH2CON H2 COOCH2COCH3 -COOCH2COOCH=CH2 -G OON = CHCH3 -COoN-C, (CH3)2 COOC-CHCOCH3 -COOCH2CH2B r -COOCR,0M2CN COOCH2CH2N-CHs C1” CH.

Typical vinylsulfonyl group or its precursor functional group that can be used in the synthesis of polymers having structural repeating units represented by any of the general formulas (1) to (3) above. A method for synthesizing saturated monomers is described in JP-A-59-171849.

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 mole percentages, and R1R' represents the following substituent.

A-1x=92, y=8, z=O R= Coo CH2CH20COCl2cH2so□
ct+=cH1A-2x=92, Y=8.2=0 R= C0NHCH□NH(:0CH2CH2so□C
) I= CH2A-3x=80, y=8, z=12 R= C0NHCH□NHCOCH2CH2SO2CH
= C12R' = CONH2C(CH3) 2 C
:H2C0CH3A-6x=92, y=a, z=O A-7x=9.2, y=8, 2=0 R= C0NHCH2NHCOCH2CH2SO2CF
12 CH2CIA-+3 x=80. y=8,
z=12R= C0NHCH2NHCOCH3C823
02CH2CI2 CIR' = CON+(2C(
CH3) 2 CH2C0CR3R' = CO
N(CH3)2 A-12x = 92, y=s, 2=0R=CO
OC: H2CH2OC0CH25O□CH=CI (2 By the way, in JP-A-59-17189, a polymer using an acrylamide component instead of the methacrylamide component of the present invention was filed, where x, y, and z are It is a molar percentage, and R1R' represents the following substituent.

B-1x=92, y=8. z=0 R=C0OCR2G)120CO(:R2CH2SO□
CH=CI(2B-2x=92, Y=8.2=0 R= C0NHCH2NHCOCH2CI2so, c
o=CH2B-3x=80, y=s, z=j2R=
C0NHCH2NHCOCI(2CH25O2(:l
= CH2R' = C0NH2C(CHri) 2 C
H2C0CR3B-5x=92, y=8. z=0
B-7x=92, y=13, 2=0 R=C0NHCH2NHCOCH2CH2SO2CH2
CH2CIB-8x=80, y=8, z;12R=
C0NHCH2NHC:0CH2CH2SO2(1:R
2CH2CIR' = C0N82C(CH3)
2 CF+2 (:0CH3R' = C0N(
CH3) 2B-12x = 92, y= 8, 2
=0R= COOCH2CH20COCH2SO2CH
= 82 In the present invention, methacrylamide copolymers function as structural support components of electrophoretic media, and aqueous gels of these methacrylamide copolymers are
The desired copolymer components are prepared by crosslinking polymerization in the presence of water. It is preferable to use a crosslinking agent during this crosslinking polymerization.

The crosslinking agent used to obtain the methacrylamide copolymer aqueous gel of the present invention includes two or more nucleophilic groups (
For example, amino groups, phenolic hydroxyl groups, sulfinic acid groups, thiol groups, combinations of amino and thiol groups)
Examples include compounds having in the same molecule.

Examples of the crosslinking agent having an amino group as a nucleophilic group include ethylenediamine, 1,3-propanediamine, 1,
5-pentanediamine, hexamethylenediamine, diethylenetriamine, triethylenetetramine, propylenediamine, di(aminomethyl)ether, 1,4-diamino-2-butene, 1,8. -Diamino-4-(aminomethyl)octane, xylylenediamine, phenylenediamine, polylysine, polyethyleneimine, gelatin, etc.

Examples of the crosslinking agent having a phenolic hydroxyl group include hydroquinone, bisphenol A, and bisphenol sulfone.

Examples of the crosslinking agent having a sulfinic acid group include 1°4-butanedisulfinic acid and benzenedisulfinic acid.

Examples of crosslinking agents having a thiol group include thioethanolamine and p-aminothiophenol.

Examples of compounds having an amino group and a thiol group include cystiamin.

The amount of the crosslinking agent to be added is preferably such that it can be gelled, that is, 0.5 to 5 equivalents to the reactive groups in the polymer of the present invention.

Next, synthesis examples of A-1, A-2, A-7 and A-8, which are particularly preferable polymers among the above polymers, will be shown.

[Synthesis Example 1] 60ml of N,N-dimethylformamide was added to the synthesis reaction ensemble of poly-2-[3-(vinylsulfonyl)propionyloxy]ethyl acrylate-comethacrylamide (A-1). - 14.5 g of -chloroethylsulfonyl)propionyloxy]ethyl acrylate and 39.0 g of methacrylamide were added, degassed with nitrogen gas, heated to 60°C, and heated to 2.2'
-Azobis(2,4-dimethylvaleronite, ryl)
[CAS Registry No. 4419-11
-8]0.4g was added, and heating and stirring were continued for 2 hours. After that, 0.20 g of 2,2'-azobis(2,4-dimethylvaleronitrile) was added, and after continued heating and stirring for 2 hours, it was cooled to 5°C, and 12 g of sodium carbonate and 4.9 g of triethylamine were added. , stirring was performed for 1 hour.

After further stirring at room temperature for 2 hours, the reaction solution was put into a cellulose tube, dialyzed for 2 days, and then lyophilized to obtain 4
6 g of a white polymer (corresponding to A-1 above) was obtained (yield 90%), the vinyl sulfonyl group content of this polymer was 0.
．． It was 93xlO-' equivalent/g.

[Synthesis Example 2] Synthesis of poly-N-([3-(vinylsulfonyl)propanamidocomethyl)acrylamide-comethacrylamide (A-2) In a 200 mM reaction vessel, N-[3-(2-chloroethyl 5.65 g of sulfonyl)propanamide methylcoacrylamide, 15.3 g of methacrylamide and 80 ml of 50% methanol aqueous solution
was added, heated to 60°C with stirring, 0.1 g of 2,2'-azobis(2,4-dimethylvaleronitrile) was added, and after another 30 minutes, o, Ig of the same was added to give 2.
Heating and stirring were continued for an hour. Thereafter, the mixture was cooled to about 10°C with ice water, and a solution of 2.5 g of triethylamine dissolved in 80 rnl of methanol was added, stirring was continued for 2 hours, and the reaction solution was poured into 1 fL of acetone without stirring. 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 Xio-coequivalents/g.

[Synthesis Example 3] Synthesis of poly-N-([3-(2-chloroethylsulfonyl)propanamidocomethyl)acrylamide-comethacrylamide (A-7) In a 500 mM reaction vessel, N-[3-(2 -chloroethylsulfonyl)propanamide 10.3 g of methylcoacrylamide, 18.7 g of methacrylamide and 50%
Add 160 ml of methanol aqueous solution and reduce to 60 ml while stirring.
The mixture was heated to 0.degree. C., 0.2 g of 2.2.degree.

The reaction solution was put into a cellulose tube and dialyzed for 2 days, and 25 g of a white polymer (corresponding to A-7 above) was obtained by freeze-drying (yield: 85%). The chloroethylsulfonyl group content of this polymer was 0.95 x lO-' equivalent/g.

[Synthesis Example 4] Copoly-N-([3-(2-chloroethylsulfonyl)propanamide methylcoacrylamide, -methacrylamide-N-(1゜1-dimethyl-3
Synthesis of -oxbutyl)acrylamide (A-8) In a 500 ml reaction vessel, 10.3 g of N-[3-(2-chloroethylsulfonyl)propanamidomethylcoacrylamide, 35.0 g of methacrylamide, N-(
1,1-dimethyl-3-oxobutyl)acrylamide (diacetone acrylamide)tt-3,H and 50
Add 160 ml of % methanol aqueous solution and add 160 ml of methanol aqueous solution to
The mixture was heated to 0° C., 0.2 of 2,2′-azobis-(2,4-dimethylvaleronitrile) was added, and 1 hour later, 0.2 g of the same was added, and heating and stirring was continued for 2 hours. The reaction solution was put into a cellulose tube, dialyzed for 2 days, and 45 g of white poly-7- (corresponding to A-8 above) was obtained by freeze-drying.
was obtained (yield 80%).

The content of tetraloethylsulfonyl groups in this polymer paste is 0.8
It was xlO-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 1,000,000, and by selecting the molecular weight, the electrophoresis medium forming solution (
The viscosity of the gel-forming liquid (hereinafter referred to as gel-forming liquid) can be adjusted. A gel electrophoretic membrane can be formed by a known method such as casting or coating this gel-forming solution on a horizontally placed support.

The electrophoresis medium (hereinafter also referred to as gel medium) of the present invention preferably contains an anionic surfactant as a denaturing agent. Particularly when the analysis sample is a protein or a complex protein (eg, riboprotein, glycoprotein, etc.), it is often preferable or essential to include an anionic surfactant. Of course, anionic surfactants may not be included in the gel medium.

Specific 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 inexpensive and easy to use. Among the alkyl sulfates, dodecyl sulfate (sodium salt, potassium salt, lithium salt, etc.) is preferred, and sodium dodecyl sulfate (SO3) is particularly preferred.
is most preferred. By incorporating an anionic surfactant, particularly SDS, into the gel medium of the present invention, it becomes possible to separate proteins or complex proteins and measure their molecular weights. The content of the anionic surfactant as a denaturing agent is about 0.05 w/v% to about 2.0% w/v of the gel forming solution. Ow/v%
, preferably about 0, 1. w/v% to approx. 1.5w
/v% range.

The surfactant that can be added to the gel medium of the present invention is described in detail in Japanese Patent Application No. 145480/1983.

The gel medium of the present invention can contain a pHl1 buffer. The buffer can be appropriately selected from known buffers capable of buffering to a pH value within the range of 2.5 to 10.0, depending on the sample to be electrophoretically analyzed.

As a buffering agent that can be used, Nippon Kagaku Yosen "Chemistry Handbook Basic Edition" (Tokyo, Maruzen @ Published in 1966) 1312-1
320 pages: “Latest Electrophoresis Methods” edited by Aomai and Mizuben (Tokyo, Tokoyo Shoten, published 1973, pages 320-322;
r Data for Biochemical Re
5earch J (R.

M. C. Dawson et al., ed., 2nd edition, 0
xford at the C1arendon Pre
ss, published in 1969) 476-508 pages;
r Biochemistry J 5.467
(1966), rAnalytical Bio
chemistry J 104.300-310 (
1980) and others.

Specific examples of buffering agents include buffering agents containing barbiturates,
Tris(hydroxymethyl)aminomethane (T r i
s) [CAS Registry No. 77
Buffer containing -86-11, buffer containing phosphate, buffer containing borate, buffer containing acetic acid or acetate, buffer containing citric acid or citrate, containing lactic acid or lactate Buffer, buffer containing glycine, N,N-bis(2-hydroxyethyl)glycine (Bicine),
N-2-Hil(loxhetilbiverazine-N', -2-
Hydroxypropane-3-sulfonic acid (HEPPSO)
or a salt thereof, N-2-hydroxyethylpiperazine-
Examples include N'-3-propanesulfonic acid (EPPS) or a salt thereof, N-[tris(hydroxymethyl)]-]3-aminopropanesulfonic acid TAPS) or a salt thereof, and the like. Specific examples of preferred buffers include potassium dihydrogen phosphate-disodium hydrogen phosphate, T
ris-sodium borate, Tris-sodium borate-EDTA ・2Na salt, Tris-citric acid, harbital sodium-sodium acetate, parbital sodium-hydrochloric acid, parbital-sodium barbital, acetic acid-sodium acetate, lactic acid-sodium lactate , citric acid-disodium hydrogen phosphate, B i c i n
e, HEPPSO1HEPPSO sodium salt, EPP
Examples include S, EPPS sodium salt, TAPS, TAPS sodium salt, and the like.

Preferably, the gel medium of the present invention further contains a water-soluble synthetic polymer compound. This synthetic polymer compound is
About 0.1% to 1% by weight based on the total weight of monomer and crosslinker
00% by weight, preferably 2% to 50% by weight.

The molecular weight of the above water-soluble synthetic polymer compound is 1
A range of 10,000 to 1,000,000 is preferred.

As a preferable example of the water-soluble synthetic polymer, addition polymerization type or condensation polymerization type water-soluble poly7- can be used. Specific examples of addition polymers include nonionic water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide. Further, specific examples of condensation polymers include nonionic water-soluble polyalkylene gellicols such as boethylene glycol and polypropylene glycol. Among these, polyethylene glycol and polyacrylamide are particularly preferred. By adding this polymer, the wet gel film becomes plastic and its brittleness is improved. Moreover, even in the state of a dry gel film, the plasticizing effect acts more effectively, and brittleness such as cracks is improved.

Preferably, the gel medium of the invention further contains agarose. The type of agarose is not particularly limited, and any of low electroosmotic, medium electroosmotic, and high electroosmotic agaroses can be used. As examples of agarose that can be used, agaroses disclosed in publications such as JP-A-55-5730, JP-A-55-110946, and Japanese Patent Application Publication No. 57-502098 can also be used. The amount of agarose added is about 0.2w/v% to about 2w based on the volume of the gel composition containing monomers and crosslinking agent.
/v%, preferably from about 0.3 w/v% to about 1.2
selected from the w/v% range. In a composition containing agarose, it is possible to control the solution viscosity to an appropriate value by changing the solution temperature.

As mentioned above, the electrophoresis medium of the present invention comprises a monomer typified by acrylamide, a difunctional allyl (aIlyl)
) compound or an acrylic compound (crosslinking agent), a water-soluble polymer, and agarose are obtained by crosslinking and polymerizing a monomer and a crosslinking agent in a substantially uniform aqueous solution, and from the monomer and crosslinking agent. It is assumed that the water-soluble polymer and agarose (if agarose is used together) are substantially dispersed in the three-dimensional cross-linked polymer formed, and the polymer chains of the latter two are entangled with the three-dimensional cross-linked polymer. be done.

The gel medium of the present invention can contain an antioxidant, if necessary. As the antioxidant, various compounds known 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 of the present invention for the purpose of wetting and improving the electrophoretic image. The content of the polyol compound is selected from the range of about 0.1 w/v% to about 40 w/v%, preferably about 0.5 w/v% to about 40 w/v%, based on the volume of the gel medium. . Among these, glycerin is particularly preferred. By adding polyol, it is possible to prevent the gel medium from drying out due to extreme water evaporation during storage, and also improve the physical properties of the gel medium, such as preventing brittleness caused by extreme drying and preventing cracking. There is an advantage in being able to do so.

When used as a gel medium or membrane of the present invention,
A gel layer or a gel film can be formed by applying a gel-forming liquid by a known method onto an electrically insulating support having a smooth wood-philic surface and then crosslinking the gel-forming liquid. .

The support may be a glass plate, a wood-loving polymer, or a polymer whose surface has been made hydrophilic by a known surface treatment (e.g., polyethylene terephthalate, polycarbonate of bisphenol A, polyvinyl chloride, vinylidene chloride/vinyl chloride copolymer, polymethyl Examples include molded products such as plates or sheets of methacrylate, polyethylene, polypropylene, cellulose acetates, cellulose acetate propionate, 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 5 electrolytic etching, etc. can be applied. can.

When the gel-forming liquid is crosslinked on the surface of the support, the gel-forming liquid can be further covered with a cover film, sheet, plate, or the like. The cover film, sheet, or plate used for this purpose may be made of the same material as the support. The thickness of these cover films and the like is 300 pm or less, with a practically preferred range of about 4 pm to about 200 pm, and a particularly preferred range of about 44 m to about 1100 JL.

It is believed that by using the electricity-like effect medium of the present invention, a complicated oxygen removal operation can be omitted and it is possible to mass-produce an electricity-like effect medium with high physical strength.

Examples and comparative examples of the present invention are shown below.

[Example 1 and Comparative Example 1] A flat, smooth glass plate was held horizontally in the air, and the copolymer gel composition liquid (gel forming liquid) listed in Table 1 was applied thereto to a thickness of about 400 JLm. The time required to complete gelation (gelation time) was measured.

Next, the compressive elastic modulus of the gel film formed on the glass plate was measured. The results of gelation time and compressive modulus are summarized in Table 2.

Table 1 Acrylamide ----11,87g Polymer
A-L A-28-I B-2-S D S
0.10g 0.10g 0.1
0g 0.10g 0.10g water
(Add water to make 100mJL) N, N, N', N
'-Tetramethylethylenediamine 33 Note: Weight of each polymer is 11.87 g; Table 2 Atmosphere and time 1.4 1.4 2.1 3.0 Soaked at 5 cm pressure without gelation: 1. 5 4.1 2.7 2
．． 9 -- Sample numbers 1 and 2 (samples according to the present invention) both have a short gelation time of 1.4 minutes, and also have large compressive moduli of 3.5 N/crn' and 4.1 N/cnf, respectively. On the other hand, it can be seen that sample number 3.4 (comparative example) had a long gelation time and a small compressive elastic modulus.

Sample 5 (gel-forming liquid containing no polymer) was prepared in air (
No gel film was formed in the presence of oxygen).

Similar results were also obtained when a thiol compound was used instead of 1,3-propanediamine.

[Example 2 and Comparative Example 2] A glass plate cell having a 400 gm thickness space was created using two planar smooth glass plates and a 400 gm thick spacer plate, and the gel described in Table 1 was formed in this cell. Solutions 1-5 were injected to form a gel medium under oxygen exclusion.

Standard proteins were subjected to electrophoresis using these gel media. As a standard protein, cytochrome C1 (molecular weight 1
2400), chymotrypsinogen A (molecular weight 2500
0), ovalbumin (molecular weight 45,000), and bovine serum albumin (molecular weight 67,000) were used.

Staining was done with 0.1% Coomasie Blue (Coomasie B).
The gel membrane was immersed in R-250.

Normal electrophoresis patterns were obtained for all sample numbers 1 to 5.

Note that when the same gel formulation as Sample No. 5 in Table 1 was used, the gel medium could be produced only under oxygen-blocked conditions, and the obtained electrophoresis pattern was normal.

[Effects of the Invention] The electrophoresis medium of the present invention can be formed even in the presence of oxygen, the time required for gelation is short, the physical strength of the gel medium is improved, and there are no problems with electrophoretic properties. Therefore, the present invention can provide a high-performance electrophoresis medium in a convenient container.

Patent Applicant Fuji Photo Film Co., Ltd. Agent Patent Attorney Yasushi Yanagawa Procedural Amendment January 28, 1988 1986 Patent Application No. 298527 2 Name of the invention Electrophoresis medium 3 Person making the amendment Relationship to the case Patent applicant name (520) Fuji Photo Film Co., Ltd. 4, agent address Miiya Yotsuya Building 8, 2-14 Yotsuya, Shinjuku-ku, Tokyo
Level 6, number of inventions increased by amendment None 7,
``Detailed Description of the Invention'' column of the specification to be amended.

8. The "Detailed Description of the Invention" column of the description of amendments will be amended as follows.

Note 1) "A flat, smooth glass...gel medium was formed." on page 47, lines 2 to 6 of the specification.
A gel medium was prepared using the same gel formulation as Sample Nos. 1 to 4 in Comparative Example 1. ” he corrected.

2) “Sample numbers 1 to 5J” on page 47, line 15 of the specification
"Correct as sample numbers 1 to 41.

that's all

Claims (1)

[Claims] 1. Cross-linked polymerization in the presence of water, with general formulas (1) to (3)
) An electrophoresis medium comprising an aqueous gel containing a methacrylamide copolymer having a constituent repeating unit represented by any of the following. \General formula (1)\ ▲Mathematical formulas, chemical formulas, tables, etc.▼ [In general formula (1), R^1^1 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; Q^ 1 is -COO-, ▲There is a mathematical formula, chemical formula, table, etc.▼, or an arylene group having 6 to 10 carbon atoms, Li is -COO-, ▲There is a mathematical formula, chemical formula, table, etc.▼ bond A divalent group containing at least one of the following and having 3 to 15 carbon atoms, or -O-, ▲Numerical formula,
There are 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 50 to 99, y^1 is 1 to 50, z^1 is the residual value (z^1 may be 0)];\General formula (
2)￥ ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In general formula (2), 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 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- (wherein R is an alkylene group having 1 to 6 carbon atoms or an arylene group having 6 to 12 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; x^2, y^ 2 and z^2 represent molar percentage, x
^2 is 50 to 99, y^2 is 1 to 50, z^2 takes the residual value (z^2 may be 0)];\ General formula (
3)￥ ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the general formula (3), R^3^1 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; L^3 is 1 is a divalent linking group having 20 carbon atoms from is a divalent group; x^3, y^3 and z^3 represent molar percentages, x
^3 is 50 to 99, y^3 is 1 to 50, z^3 is the residual value (z^3 may be 0)]; m is 0 or 1] 2. Methacrylamide 2. The medium according to claim 1, which is a polymethacrylamide aqueous gel electrophoresis medium obtained by cross-linking polymerizing a cross-linking agent and a cross-linking agent in the presence of water, further comprising a water-soluble polymer and agarose. 3. The medium according to claim 1 or 2, further comprising an anionic surfactant as a modifier. 4. The medium according to claim 3, wherein the anionic surfactant is an alkyl sulfate. 5. The medium according to claim 4, wherein the alkyl sulfate is sodium dodecyl sulfate. 6. With respect to the total weight of the methacrylamide compound and the crosslinking agent, the crosslinking agent is in the range of 2 wt% to 30 wt%, the water-soluble polymer is in the range of 2 wt% to 100 wt%, and the agarose is the polymethacrylamide type. 0.2wt/v% to 2wt/v% relative to the volume of the aqueous gel
The medium according to claim 2, which is contained within the range of . 7. The modifier is 0.05 wt/v% to 2.0 wt/v with respect to the volume of the polymethacrylamide-based aqueous gel.
% of the medium according to any one of claims 3 to 5.