JPS61296258A - Electrophoretic medium material and its preparation - Google Patents

Abstract

PURPOSE:To attain to enhance the adhesiveness between a support and a medium layer, by forming a three-layered structure consisting of a plastic support layer, an adhesive layer and an electrophoretic medium layer comprising a polyacrylamide type aqueous gel. CONSTITUTION:A plastic support layer and an electrophoretic medium layer comprising a polyacrylamide type aqueous gel are adhered by an adhesive layer formed of an amido group-containing grafted derivative of a methyl methacrylate macromonomer to form a three-layered structure. In this structure, said three-layered structure becomes hard to separate even by various operations in the dyeing process of the medium layer.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention 1] The present invention relates to an electrophoretic medium material and a method for producing the same. The present invention relates to suitable electrophoretic media materials and methods of manufacturing the same.

[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 [Experimental Electrophoresis Methods (Revised 5th Edition)]
” (Bunkodo, published in 1975).

“Latest Electrophoresis Methods” edited by Aomizu and Mizuben (Yokouse Bookstore).

(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 been used as a membrane or sheet for electrophoresis for a long time, but as mentioned above, agarose membranes and polyacrylamide gel membranes have recently been used from the viewpoint of performance. Acrylamide gel membranes are currently most commonly used.

Polyacrylamide gel membranes are produced 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 has been obtained.

Anionic surfactants are often added as denaturing agents when producing polyacrylamide gel membranes, but in the production of gel membranes for protein analysis, the amount of denaturing agent required is small, so it is not necessary to add denaturing agents to wet gel membranes. The modifier can be impregnated into the gel film by a method of applying an aqueous modifier solution, a method of immersing the gel film in an aqueous modifier solution, or the like.

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, the polyacrylamide gel film must be prepared in a state where oxygen is blocked. For this reason, polyacrylamide gel membranes are generally produced in cells consisting of two glass plates (a certain space, e.g. 0.3 mm - 1 m).
It is formed by injecting a gel-forming liquid into (m), blocking oxygen, and cross-linking and polymerizing it to form a gel.

However, the work of forming a gel film between two glass plates has disadvantages such as the glass being easily broken and heavy, and the gel film sandwiched between glass plates being inconvenient to carry and difficult to handle. It is extremely difficult to mass produce it.

Furthermore, in the past, horizontal or vertical slab electrophoresis was performed using a polyacrylamide gel membrane sandwiched between glass plates under predetermined conditions for a fixed time period (m = m), and then the gel membrane was stained (e.g., 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 wet gel membrane, the gel membrane is damaged during this staining process. It peels off more easily than a glass plate, and requires highly skilled techniques.

In order to solve the above-mentioned difficulty in handling, it is desired to develop a polyacrylamide gel membrane provided on a light plastic support instead of a glass support. Preferred materials for plastic supports used for this purpose include plastics that are easy to handle and have excellent performance, such as polyethylene terephthalate (PET), but these plastics are generally hydrophobic. Therefore, there is a problem of poor adhesion between the gel film and the support. Further, even if the surface of the hydrophobic plastic sheet is made hydrophilic, or even if a hydrophilic plastic sheet is used, the adhesion between the gel film and the support does not necessarily reach a satisfactory level.

[Summary of the Invention] Therefore, the present invention provides an electrophoresis medium material comprising a plastic support layer and an electrophoresis medium layer (polyacrylamide gel), which is easy to handle and has a high electrophoresis medium layer in a wet state. The object of the present invention is to provide an electrophoretic medium material with excellent adhesion between a layer and a support, and a method for producing the same.

The present invention also provides an electrophoresis medium material and a method for producing the same, in which the electrophoresis medium layer (polyacrylamide gel membrane) does not peel off from the support during the dyeing process after electrophoresis or the drying process after the staining process. Its purpose is also to provide.

The present invention consists of an electrophoretic medium material having a three-layer structure in which the following layers are sequentially laminated: [I] Plastic support layer; [n] Grafting of methyl methacrylate macromonomer containing an amide group. An adhesive layer formed from a derivative; and.

[III] A polyacrylamide-based aqueous gel electrophoresis medium layer formed by cross-linking polymerization of an acrylamide-based compound and a cross-linking agent in the presence of water.

The electrophoretic medium/material of the present invention has a three-layer structure in which the support layer and the electrophoretic medium layer are bonded together by an adhesive layer formed from an amide group-containing grafted derivative of methyl methacrylate macromonomer as described above. The three-layer structure is difficult to separate even during various operations in the dyeing process of the electrophoresis medium layer (polyacrylamide gel membrane) described above, which is very advantageous in one operation. Furthermore, the electrophoretic medium material of the present invention can be obtained by forming a layer of an amide group-containing grafted derivative of methyl methacrylate macromonomer on a horizontally placed support, and then forming an electrophoretic medium layer thereon. Since it is also possible to manufacture the material using a method, some of them can greatly contribute to the mass production of electrophoretic media materials.

The above-mentioned electrophoretic medium material is prepared by providing an intermediate layer containing an amide group-containing grafted derivative of methyl methacrylate macromonomer on a plastic support, and then applying an acrylamide compound and a crosslinking agent on the intermediate layer. It can be manufactured by utilizing a method of forming a polyacrylamide-based aqueous gel electrophoresis medium layer by cross-linking polymerization in the presence of water.

[Detailed Description of the Invention] Various plastic sheets can be used as the support for the electrophoresis medium material of the present invention. As this plastic sheet, one formed of any plastic can be used. Examples of preferred plastic sheets include hydrophilic polymers or polymers whose surfaces have been made hydrophilic by known surface treatments (e.g., polyethylene terephthalate, bisphenol A polycarbonate,
Examples include sheets (including films and plates) of polyvinyl chloride, vinylidene chloride/vinyl chloride copolymer, polymethyl methacrylate, polyethylene, polypropylene, cellulose acetates, cellulose acetate propionate, etc.). As a treatment for making the surface of the plastic sheet 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.

However, it is not essential to make the surface of the support a woody material, and the above-mentioned plastic sheet can be used as it is as the support.

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

The adhesive layer is a layer formed from an amide group-containing grafted derivative of methyl methacrylate macromonomer. A macromonomer is generally defined as a polymerizable monomer having a certain repeating unit and a relatively high molecular weight substituent, and the amide group-containing grafted derivative of methyl methacrylate macromonomer is a polymerizable It is a macromonomer containing methyl and methacrylate group derivatives in its molecule. The amide group-containing grafted derivative of methyl methacrylate macromonomer is a monomer containing one amide group-containing ethylenically unsaturated double bond, preferably a kind of amide group-containing acrylic acid or methacrylic acid compound. Alternatively, it is a compound obtained by treating with two or more kinds of monomers to initiate graft copolymerization with the monomers at the methyl methacrylate group end of the methyl methacrylate macromonomer. Examples of amide group-containing acrylic acid compound monomers include acrylamide, N-methylacrylamide, N
Examples of amide group-containing methacrylic acid compound monomers include methacrylamide, N-methylmethacrylamide, and the like.

Synthesis examples of typical amide group-containing grafted derivatives of methyl methacrylate macromonomers used in the present invention are described below. In addition, in each synthesis example, 1 part' and 1 part
%" means r parts by weight J and "% by weight", respectively.

[Synthesis Example 1] A mixed solvent of 17.5 parts of acetone and 82.5 parts of toluene was charged into a glass flask equipped with a stirrer, a reflux condenser, a dropping funnel, a thermometer, and an N2 gas inlet, and while introducing N2. Methyl methacrylate (hereinafter MMA) was added under reflux.
) 100 parts, 3.2 parts of thioglycolic acid (hereinafter abbreviated as TGA) as a chain transfer agent, and azobisisobutyronitrile (hereinafter abbreviated as AIBN) as a polymerization initiator.
Polymerization was carried out by continuously dropping 3 parts of the mixed solution over 3 hours. After that, the polymerization was completed by heating for 2 hours.
N-hexane was added to a portion of the 0 reaction solution obtained from the solution of the polymer having the following structural formula [1] to form a precipitate, and the precipitate was dried under reduced pressure and the acid value was measured, and it was found to be 0. 340
mg equivalent m/g.

After distilling off a portion of the acetone from the above reaction solution, 0.5% of triethylamine (Tokuko), 200 ppm of hydroquinone methyl ether (polymerization inhibitor), and 1.3 times the molar amount of glycidyl methacrylate relative to the acid were added. In addition,
The reaction was carried out at a reaction temperature of 110° C. for 4 hours. The reaction rate determined from the decrease in acid value was 96%.

The above reaction solution was poured into n-hexane in an amount equal to 10 inoculations to form a precipitate, and this precipitate was dried under reduced pressure at 80°C to obtain 95 parts of a macromonomer of the following structural formula [II]. The polystyrene equivalent molecular weight of this macromonomer by gel permeation chromatography (GPC) is 2840.
(number average) and 6.470 (weight average). Also, the hydroxyl value was 0.350 mg equivalent/g. [Synthesis Example 2] Synthesis of comb-shaped graft polymer In the same manner as Synthesis Example 1, 30 parts of macromonomer obtained in Synthesis Example 1, 50 parts of MMA, 20 parts of diacetone acrylamide
1, 200 parts of methyl ethyl ketone and 3.0 parts of azobisisobutyronitrile (hereinafter abbreviated as AIBN) were charged, and the mixture was reacted for 800 hours under reflux (approximately 80° C.) by introducing N2 gas.

This reaction solution was poured into to @ amount of n-hexane to precipitate it, and it was dried under reduced pressure at 80°C to obtain 94 parts of a graft copolymer (P-1). The obtained graft copolymer showed a single peak by GPC. The number average molecular weight was approximately 19,000.

By the same operation, two types of comb-shaped graft polymers (P-1
and P-3) were synthesized. Table 1 shows each comb-shaped graft colorimer.

Table 1 number in the margin below Copolymerization monomer Average molecular weight of comb-shaped graft polymer Yield P-1 macromonomer 30 parts Approximately 19.000 92
Parts MMA 50 parts (A) 20 parts P-2 macromonomer 30 parts Approximately 22.000 92
Part MMA 30 parts (A) 40 parts P-3 macromonomer 30 parts Approximately 12.000 95
Part MMA 30 parts (B) 40 parts Note) (A) Nidiacetone acrylamide (B): N, N
- Dimethylacrylamide MMA: Methyl methacrylate These amide group-containing grafted derivatives of methyl methacrylate macromonomers may be used alone or in combination, and may be used in combination with other highly concentrated derivatives as long as the amount is within 50% by weight of the layer-forming components. It can contain molecular substances and additives. Examples of such polymeric substances and additives include diacetylcellulose, nitrocellulose, polyvinyl alcohol,
Examples include polymers such as polyacrylamide, polymethyl methacrylate, and polyvinylidene chloride, and polyol compounds such as glycerin.

The adhesive layer formed from the amide group-containing grafted derivative of methyl methacrylate macromonomer can be provided on the surface of the support using a known coating method.

That is, when the macromonomer derivative is water-soluble or wood-philic, an aqueous solution thereof or a water/organic solvent mixture solution containing water as the main component is applied onto a support by a known method and dried. A method of forming an adhesive layer can be used. When the macromonomer derivative is hydrophobic and water-insoluble, a solution thereof in an organic solvent or
An organic solvent/water mixed solvent solution containing an organic solvent as the main component,
A known method can be used in which the adhesive layer is formed by coating on a support and drying.

Examples of organic solvents that can be used include acetone,
Ketones such as methyl ethyl ketone; Alcohols such as methanol and ethanol; N.

Examples include N-dimethylformamide; dimethyl sulfoxide; ethers such as dimethyl ether and dioxane.

The adhesive layer formed from the amide group-containing grafted derivative of the methyl methacrylate macromonomer is desirably formed from the amide group-containing grafted derivative of the methyl methacrylate macromonomer alone or from a composition containing 80% by weight or more of the derivative. In particular, it is desirable that the layer be formed only from an amide group-containing grafted derivative of methyl methacrylate macromonomer.

The thickness of the adhesive layer after drying is about 0.1 gm to about 3 μm,
Preferably, the range is about 0.21 Lm force and about 2 gmc7).

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

Polyacrylamide gel membrane is an aqueous gel obtained by dissolving or dispersing an acrylamide compound and a crosslinking agent in water as an aqueous solution or dispersion to prepare a gel forming liquid, and then crosslinking and polymerizing both in the liquid. In this specification, unless otherwise specified, dissolution (in water) and dissolution (in water)
The term ``dissolution (in water)'' includes both dispersion, and the term ``aqueous solution'' includes both an aqueous solution and an aqueous dispersion. Also, the term solvent or dispersion medium includes a mixture of an organic solvent and water that may be added as desired. do.

Acrylamide compounds that can be used for producing polyacrylamide gel membranes include acrylamide, N
-Methylacrylamide5. Examples include acrylamide homologues such as N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide, and diacetone acrylamide, and mecrylamide-based compounds such as mecrylamide, and these compounds may be used alone or in combination of two or more types. be able to. Among these acrylamide compounds, acrylamide is the most preferred;
It is also preferable to use acrylamide and one or more other acrylamide compounds in combination.

As a crosslinking agent, “Electrophoresis (Electr
ophoresis) J 1981.2.220-2
Known compounds (one type or a combination of two or more types) described in 28 etc. can be used. Specific examples of crosslinking agents include N,N'-methylenebisacrylamide (BIS),
N,N'-propylene bisacrylamide (PBA),
di(acrylamidomethyl)ether (or N, N'
-oxydimethylene acrylamide); 1,2-diacrylamide ethylene glycol; 1,3-diacryloyl ethylene urea; ethylene diacrylate (EDA)
;N,N'-diallyltartardiamide (N,N'-
dial+71tartardiamide, D A
T D ); and N.

N”-bisacrylylcystamine (N, N'-bis
acrylylcystamine, BAC)
Examples include iso-bifunctional compounds.

The crosslinking agent may be used in an amount ranging from about 0.1% to about 30%, preferably from about 0.5% to about 10% by weight, based on the total weight of monomer and crosslinking agent. .

As for the gel concentration, Nis Hegerten (S.

Arch, Biochem, Biop
hys,) J 1 (Addendum).

147 (1982),
The amount of monomer and crosslinker is from about 3 w/v % to about 3% relative to the volume of the gel medium consisting of monomer, crosslinker and water.
It is preferably used within the range of 0 w/v%.

The electrophoresis medium material of the present invention is mainly used to advantageously analyze proteins or complex proteins (e.g., riboproteins, glycoproteins, etc.), and the electrophoresis medium layer contains an anionic denaturant as a denaturing agent. It is preferable or essential to include an anionic surfactant when the analytical sample is a protein or complex protein (e.g. lipoprotein, glycoprotein, etc.) It goes without saying that the gel medium layer may not contain an anionic surfactant, which contains a large amount of anionic surfactants. For example, a gel media layer containing no anionic surfactant is D
It can be used for purposes such as genetic disease diagnosis based on NA fragment analysis or DNA structure analysis using restriction enzyme digestion.

By including an anionic surfactant in 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. Among the sulfates, dodecyl sulfate (sodium salt, potassium salt, lithium salt, etc.) is preferable, and sodium dodecyl sulfate (SDS) is the most preferable.By incorporating SDS into the gel medium layer of the present invention, protein or complex Efficient separation of proteins and measurement of their molecular weight becomes possible.

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 it ranges from about 0.1 w/v% to about 1.5 w/v%.

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

A water-soluble polymer is optionally added to the polyacrylamide gel membrane. 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.

Among these water-soluble polymers, polyacrylamide and polyethylene glycol are preferred.

Preferably, the molecular weight of the water-soluble polymer is in the range of about 10,000 to about 1 million, the water-soluble polymer is in the range of about 2% to about 100% by weight, based on the total weight of monomer and crosslinker, and Preferably, it is used in a range of about 5% to about 50% by weight.

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. The polyacrylamide gel membrane preferably further contains agarose. Any agarose can be used, and any of low electroosmotic, medium electroosmotic, and high electroosmotic agaroses can be used. can. Examples of agarose that can be used include JP-A-55-5730 and JP-A-55-1109.
No. 46, JP-A No. 57-502098, and other publications disclose 7 galose and the like. The agarose is about 0.2 w/v% to about 2 w/v%, preferably about 0.3 w/v% to about 1.2 w/v%, based on the volume of the aqueous gel containing the Qi polymer and the crosslinker. used at a rate of

When the polyacrylamide gel membrane contains 7 galose, it is possible to control the viscosity of the gel to an appropriate level by changing the temperature of the gel-forming solution.
It has the advantage of being able to stop its fluidity and making it easier to mold the gel film.

The gel medium layer may also contain a pH buffer.

As a buffer, depending on the sample to be electrophoretically analyzed, P
H2, 5 to 10. It is possible to use an appropriate buffer selected from known buffering agents capable of buffering the pH value within the range of 0.

As a buffering agent that can be used, Nippon Kagakusen ``Chemical Handbook Basic Edition'' (Tokyo, Marukubi ■19)
(Published in 1873) Pages 1312-1320; Edited by Aomizu and Ben Mizu, "Latest Electrophoresis Methods" (Tokyo, Tokonyo Shoten, Published in 1873, Pages 320-32:2; "Data for Biochemical Research"
chemical research) J (R, M, C, Datson)
External Line, 2nd Edition, Oxford at the C1ar
Endon Press.

Published in 1969) Pages 47B-508; “Biochemistry (Bioche+5iStr7) J5
, 4B? (198B); and “Analyti Biochemistry”
cal BiochemiStr7J 104. 3
00-310 (19B0) and the like.

Examples of M buffers include Mm agents containing barbital, buffers containing tris(hydroxymethyl)aminomethane (Tris), buffers containing phosphate, buffers containing borate, acetic acid or acetates. Buffers, buffers containing citric acid or citrate, buffers containing lactic acid or lactate,
Buffer containing glycine, N,N-bis(2-hydroxyethyl)glycine (Bicine), N-2-hydroxyethylpiperazine-N'-2-hydroxypropane-
3-sulfonic acid (HEPPSO) or its salt, N-2
-Hydroxyethylpiperazine-N'-3-propanesulfone # (EPPS) or its salt, N-[Tris(
Examples include hydroxymethyl)]-]3-aminopropanesulfonic acid TAPS) or a salt thereof, and acids, alkalis, or salts that are optionally combined with any of these. Specific examples of preferred buffers include potassium dihydrogen phosphate-disodium hydrogen phosphate, Tr
is・sodium borate, TriS0 sodium borate・EDTA2Na salt, Tris・phosphoric acid, sodium parbital-sodium acetate/sodium curbital-hydrochloric acid, barbital/sodium parbital,
Sodium acetate, lactic acid, sodium lactate, citric acid-disodium hydrogen phosphate, Bicine,
HEPPSO, HEPPSO sodium salt, EPPS,
Examples include EPPS sodium salt, TAPS, TAPS sodium salt, and the like.

The electrophoretic medium layer (polyacrylamide gel membrane) in the electrophoretic medium material of the present invention is made of monomers typified by acrylamide, difunctional allyl (alyl) as described above.
lyl) compound or an acrylic compound (crosslinking agent), a water-soluble polymer, and agarose, are obtained by radical crosslinking polymerization of the monomer and the crosslinking agent in a substantially uniform aqueous solution, and the monomer and It is presumed that the water-soluble polymer and agarose are substantially dispersed in the three-dimensional cross-linked polymer formed from the cross-linking agent, and the polymer chains of the latter two are entangled with the three-dimensional cross-linked polymer.

The above radical crosslinking polymerization reaction can be carried out in the absence of molecular oxygen in the presence of peroxide and/or by a known method such as ultraviolet irradiation.

Furthermore, this crosslinking polymerization reaction can also be accelerated by heating or ultraviolet irradiation.

As a catalyst for radical crosslinking polymerization, "Electro 7, Electrophoresis J 198
1.2°213-219. 1981.2.220-2
28; Edited by Aomi and Mizuben, “Latest Electrophoresis Methods J” (197
It is possible to appropriately select and use the known low-temperature radical polymerization initiators described in the following publication (published in 2010). Specific examples of preferred radical polymerization initiators include β-dimethylaminopropionitrile (DMAPN)-ammonium peroxonisulfate mixture, N,N,N',N'-tetramethylethylenediamine (TEMED)/ammonium peroxonisulfate mixture, TEMED/riboflavin. Examples include combinations of mixtures, TEMED-riboflavin/hydrogen peroxide mixtures, and ultraviolet irradiation. The content of the radical polymerization initiator is about 0.3% to about 5% by weight based on the total weight of monomer and crosslinking agent, and preferably about 0% by weight.
．． It ranges from 5% to about 3% by weight.

The gel medium layer is formed by coating a gel forming liquid by a known method on the layer containing the above-mentioned methyl methacrylate macromonomer derivative provided on a support having a smooth surface, and then crosslinking the gel forming liquid. By polymerizing, it can be formed into layers.

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 less than 300 gm, with a practically preferred range of about 4 pm to about 200 gm, and a particularly preferred range of about 4.0 gm. gm to about Zoo, gm.

Note that the polyacrylamide gel film can also contain a polyol compound such as glycerin or ethylene glycol as a wetting agent. The content of the polyol compound ranges from about 5 w/v% to about 40% based on the volume of the gel membrane.
selected from the w/v% range. Among these, glycerin is particularly preferable. By adding a wetting agent, it is possible to prevent the gel film from drying out due to extreme water evaporation during storage, and also prevents the occurrence of brittleness caused by extreme dryness and prevents cracking. This has the advantage that the physical properties of the gel film are improved, such as preventing.

Since the electrophoresis medium layer (polyacrylamide gel membrane) in the electrophoresis medium material of the present invention has high adhesion (high adhesion force) to the support, in the normal process, the electrophoresis medium layer and the support are can always be treated as a unit. Therefore, by using the electrophoresis medium material of the present invention, it becomes possible to omit the complicated operation steps conventionally required in the electrophoresis operation of proteins or complex proteins. Media layer (
It is now possible to perform electrophoresis and staining operations with the polyacrylamide gel (@) placed on it.

Next, examples of the present invention will be shown.

[Example 1] A polyethylene terephthalate (PET) sheet (support) whose surface was made hydrophilic by ultraviolet irradiation treatment had a thickness of about 0.
A coating liquid obtained by dissolving the amide group-grafted derivative of the methacrylate macromonomer obtained in the above synthesis example in acetone to a thickness (solid content) of 57 zm (Table 2)
was applied and dried at about 110° C. to form an amide group-grafted derivative layer (adhesive layer) of the macromonomer.

Table 2 in the margin below: Composition sample of coating liquid for adhesive layer formation Macromonomer Coating liquid concentration number
Derivative (g/100ml solvent) l Synthesis Example 1 product 5g2 F-1 (Synthesis Example 2 product) 5g3 F-2 (Synthesis Example 2 product) 5g
4 F-3 (Product of Synthesis Example 2) 5 g First, the adhesiveness between the PET sheet (support) and the adhesive layer was evaluated by a cross-cut method. As a result, in all samples 1 to 4, the adhesive layer was uniformly and firmly adhered to the support, and in particular, in samples 2 to 4 of the present invention, the adhesive layer was particularly strongly adhered to the support.

On top of each adhesive layer provided on the support, 9.5 g of acrylamide, 5 g of BISo,! Ammonium belloxonisulfate (5 wt. %) 1.3mJL, TEMED
33ILi was added and molded to a thickness of 0.5 mm.
A polyacrylamide gel membrane was formed.

A comparative sample was prepared by forming a polyacrylamide gel film on the PET sheet L in the same manner except that no separate layer of the amide group-grafted derivative of the macromonomer was provided.

The obtained gel film was pressed with a finger to evaluate the adhesion between the gel film and the support. As a result, comparative sample 1 and samples 2 to 4 of the present invention had excellent adhesive properties as an adhesive layer, but the comparative sample had poor adhesive properties as compared to samples 2 to 4 of the present invention.

[Example? ] A PET sheet was formed with the same adhesive layer as in Table 2 of Example 1, and 9.5 g of acrylamide was placed on this adhesive layer.
, BI30.5g, Agarose 1600 (manufactured by Wako Pure Condiments) 0.3g, polyacrylamide 2.5g, sodium dihydrogen phosphate 12 salts 1.58g, sodium dihydrogen phosphate 2 bottles Jfi 0.33g, Oyo rJ S D S
Add ammonium sulfate (5% by weight) as a polymerization initiator to a 100mM solution containing 10g of 1.3
0.5mmc with TEMED33.1
7) Four types of samples (samples 1 to 4) were obtained by molding to a certain thickness to form a polyacrylamide gel film.

Using this gel membrane, standard proteins were subjected to electrophoresis.Then, the gel membrane was subjected to electrophoresis using 0.1% combinable blue (CoCo).
omassie Blue) R-250 (Golor
IndexConstitution Number
42880) and dyeing was performed, and the state of adhesion between the support and the gel film during this dyeing process was observed.

In Comparative Sample 1 and Samples 2 to 4 of the present invention, the gel films were completely adhered to the support throughout the dyeing process. Furthermore, after shaking the gel film vigorously for 1 hour, the adhesion state was observed. Comparative Sample 1 showed some peeling, while Samples 2 to 4 of the present invention were completely adhered. There were no problems with any of the electrophoretic properties.

[Example 3] A polyacrylamide gel film was formed in the same manner as in Example 2 on a PET sheet on which the same adhesive layer as in Table 2 of Example 1 was formed, and three types of samples of the present invention (Samples 2 to 3) were prepared. 4) was prepared. In addition, a comparative sample 1 was also prepared in the same manner as in Example 2.

After this gel film was cut along with the support, the cut section of the gel film was observed, and it was found that some peeling of the gel film was observed for comparative sample 1, whereas samples 2 to 4 of the present invention
It was found that the adhesion at the cutting edge was good in all cases, and that the entire support could be cut.

[Example 4] An amide group-grafted derivative layer of three types of macromonomers was formed on a PET support by a method similar to that described in Example 1, and the amide group-grafted derivative layer of the macromonomer was formed. Acrylamide on top 4.56
g, BISo, 24g, polyacrylamide 1.2g,
0.3 g of agarose, tris(hydroxymethyl)aminomethane [CAS Registry No. ? −
88-1] 1, 08g, boric acid 0.55g and E
Ammonium belloxonisulfate (5 wt%) 1.3 mM as a polymerization initiator was added to a 100 m solution containing 93 mg of DTA-2Na salt and 20 g of glycerin, and TEMED33.
Okibun was applied to a thickness of 1 mm, and polymerized under nitrogen gas to form a polyacrylamide gel film.

E. coli plasmid pBR-322 was transformed with restriction enzyme Asul.
When the DNA degradation pattern was examined by ethidium staining, a normal staining pattern was found.

The gel film was cut using a cutter knife to form a sample injection port. The cut was sharp. Furthermore, it was found that the separated DNA bands could be fractionated with high precision.

Claims (1)

[Claims] 1. An electrophoretic medium material comprising a three-layer structure in which the following layers are sequentially laminated: [I] Plastic support layer; [II] Grafted derivative containing an amide group of methyl methacrylate macromonomer and [III] a polyacrylamide-based aqueous gel electrophoresis medium layer formed by cross-linking polymerization of an acrylamide-based compound and a cross-linking agent in the presence of water. 2. The electrophoresis medium material according to claim 1, wherein the medium layer further contains a water-soluble polymer and agarose. 3. The electrophoretic medium material according to claim 1 or 2, wherein the medium layer further contains a modifier consisting of an anionic surfactant. 4. The electrophoretic medium material according to claim 3, wherein the anionic surfactant is an alkyl sulfate. 5. The electrophoresis medium material according to claim 3, wherein the anionic surfactant is sodium dodecyl sulfate. 6. The electrophoretic medium material according to claim 1, wherein the plastic support layer is made of polyethylene terephthalate. 7. A patent claim characterized in that the adhesive layer formed from the amide group-containing grafted derivative of methyl methacrylate macromonomer is a layer formed substantially only from the amide group-containing grafted derivative of methyl methacrylate macromonomer. The electrophoresis medium material according to item 1. 8. After providing an intermediate layer containing an amide group-containing grafted derivative of methyl methacrylate macromonomer on a plastic support, crosslinking polymerize an acrylamide compound and a crosslinking agent in the presence of water on the intermediate layer. A method for producing an electrophoresis medium material, comprising: forming a polyacrylamide-based aqueous gel electrophoresis medium layer. 9. The method for producing a medium material for electrophoresis according to claim 8, wherein the medium layer further contains a water-soluble polymer and agarose. 10. The method for producing a medium material for electrophoresis according to claim 8 or 9, wherein the medium layer further contains a modifier consisting of an anionic surfactant.