JPH0471179B2 - - Google Patents

Description

[Detailed description of the invention]

[Field of the Invention] The present invention relates to an electrophoretic medium material, and more particularly to an electrophoretic medium material suitable for use in electrophoretic analysis of biopolymer components such as proteins. . [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, a voltage is applied to both ends of the support, it is developed (moved) on or within the support, and then dyed, and this stained sample One example is an embodiment in which each component of a substance is quantitatively analyzed by measuring the optical density of the substance. For details on such electrophoretic analysis and electrophoretic membranes, please refer to "Electrophoresis Experimental Methods (Revised 5th Edition)" edited by the Electrophoresis Society (Bunkodo, published in 1975), "Latest Electrophoresis" edited by Aomoto and Nagai. Law” (Hirokawa Shoten, published in 1973), etc. In recent years, electrophoresis has been frequently used to analyze biological components, and protein analysis in particular is frequently used in biochemical tests for disease diagnosis. Filter paper has long been used as membranes or sheets for electrophoresis, but as mentioned above, agarose membranes and polyacrylamide gel membranes have recently been used from a performance standpoint, and they have particularly effective molecular sieving effects. Polyacrylamide gel membranes are currently most commonly used. Polyacrylamide gel membranes are made by combining monomers such as acrylamide with N,
It is obtained by polymerization crosslinking in the absence of oxygen using a difunctional crosslinking agent such as N'-methylenebisacrylamide. 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, the polymerization reaction for forming polyacrylamide is radical crosslinking polymerization. Crosslinking polymerization is inhibited by the influence of oxygen. Therefore, the polyacrylamide gel membrane must be prepared in a state where oxygen is blocked. For this reason, polyacrylamide gel membranes are generally produced by injecting a gel-forming liquid into a cell (certain space, e.g. 0.3 mm to 1 mm) made up of two glass plates, blocking oxygen, and allowing crosslinking polymerization to occur. It is formed by 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, after performing horizontal or vertical slab electrophoresis for a certain period of time under predetermined conditions using a polyacrylamide gel membrane sandwiched between glass plates,
The gel membrane is stained (e.g., Ponsot 3R staining, Coomassie brilliant blue G-250 staining, silver staining, etc.) and biological components are analyzed. Due to poor adhesion to the gel film, the gel film peels off more easily than the glass plate during this dyeing process, and this work 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. The preferred material for the plastic support used for this purpose is a plastic that is easy to handle and has excellent performance, such as polyethylene terphthalate (PET), but these plastics are generally It is hydrophobic and therefore has a problem of poor adhesion between the gel film and the support. Furthermore, even if the surface of a hydrophobic plastic sheet is made hydrophilic,
Alternatively, 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] Accordingly, the present invention provides an electrophoresis medium material comprising a plastic support layer and an electrophoresis medium layer (polyacrylamide gel membrane), which is easy to handle and suitable for electrophoresis in a wet state. The object of the present invention is to provide an electrophoresis medium material that has excellent adhesion between a medium layer and a support. The present invention also provides an electrophoresis medium material in which the electrophoresis medium layer (polyacrylamide gel membrane) does not peel off from the support even during a dyeing process after electrophoresis or a drying process after staining treatment. is also its purpose. The present invention consists of an electrophoretic medium material comprising a three-layer structure in which the following layers are laminated in sequence: [] a plastic support layer; [] an adhesive layer containing a cellulose derivative; and [] A polyacrylamide-based aqueous gel electrophoresis medium layer, which is formed by cross-linking polymerization of an acrylamide-based compound and a cross-linking agent in the presence of water, and contains an anionic surfactant as a modifier. The electrophoresis medium material of the present invention has a three-layer structure in which the support layer and the electrophoresis medium layer are bonded together by an adhesive layer containing a cellulose derivative as described above, Even during various operations in the dyeing process of the medium layer (polyacrylamide gel membrane), the three-layer structure is difficult to separate, 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 containing a cellulose derivative is formed on a horizontally placed support, and then an electrophoresis medium layer is formed thereon. Because it is possible to
This will greatly contribute to the mass production of electrophoretic media materials. [Detailed Description of the Invention] Various plastic sheets can be used as the support for the electrophoretic medium material of the present invention. The plastic sheet may be made of any plastic. Examples of preferred plastic sheets include:
Hydrophilic polymers or polymers whose surface has been made hydrophilic by known surface treatments (e.g., polyethylene terephthalate, polycarbonate of bisphenol A, polyvinyl chloride, vinylidene chloride/vinyl chloride copolymer, polymethyl methacrylate, polyethylene, polypropylene, cellulose) Acetates, cellulose acetate propionate, etc.) sheets (including films and plate-like materials), and the like. 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 necessary to make the support surface hydrophilic.
The plastic sheet described above can be used as is as a support. In the present invention, an adhesive layer containing a cellulose derivative is provided on the support. As the cellulose derivative, diacetylcellulose, triacetylcellulose, nitrocellulose, etc. are preferred, but other cellulose derivatives can also be used as long as they have film-forming properties. These cellulose derivatives may be used alone or in combination. Further, other polymeric substances and additives can be included as long as they are within 50% by weight of the layer-forming components. Examples of such polymeric substances and additives include gelatin, dextran, polymethyl methacrylate,
Examples include polyacrylamide, polyacrylic acid, polyvinyl alcohol, p-chlorophenol, resorcinol, glycerin, and ethylene glycol. The adhesive layer containing a cellulose derivative desirably contains 80% by weight or more of the cellulose derivative, and is particularly desirably a layer consisting essentially only of the cellulose derivative. Further, this adhesive layer may be a single layer or a composite layer, and in the case of a composite layer, the layers other than the layer containing the cellulose derivative may be other polymer layers. Examples of such other polymeric layers include layers comprising gelatin, polymethyl methacrylate, polyacrylamide, polyvinyl alcohol, polyvinylidene chloride, etc., and these polymeric layers include layers containing cellulose derivatives. It can be provided below the 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. It can be obtained by forming it as a film. In this specification, unless otherwise specified, the term ``dissolution (in water)'' includes both dissolution (in water) and dispersion (in water), and the term ``aqueous solution'' includes both an aqueous solution and an aqueous dispersion. . The solvent or dispersion medium also includes a mixture of an organic solvent and water that may be added as desired. Acrylamide compounds that can be used to produce polyacrylamide gel membranes include acrylamide, N-methylacrylamide, N,N
- Examples include acrylamide homologs such as dimethylacrylamide, N-(hydroxymethyl)acrylamide, and diacetone acrylamide, and mecrylamide-based compounds such as mecrylamide, and these compounds can be used alone or in combination of two or more types. . 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, "Electrophoresis" 1981, 2 ,
220-228, 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(acrylamidomethyl)ether (or N,N'-oxydimethyleneacrylamide). ; 1,2-diacrylamide ethylene glycol; 1,3-diacryloyl ethylene urea; ethylene diacrylate (EDA); N,N'-diallyltartardiamide (DATD); N,
N'-bisacrylylcystamine (N,N'-
Examples include bifunctional compounds such as bisacrylylcystamine (BAC). The crosslinking agent is present in an amount of about 2% to about 30% by weight, preferably about 3% by weight based on the total weight of monomer and crosslinking agent.
It can be used in amounts ranging from about 10% by weight. As for the gel concentration, S. Hjerten: "Arch.
Biochem. Biophys. 1 (Suppl.), 147 (1962). The amount used is preferably in the range of 3 w/v% to about 30 w/v%. The electrophoresis medium material of the present invention is mainly used to advantageously analyze proteins or complex proteins (e.g., lipoproteins, glycoproteins, etc.), and the electrophoresis medium layer contains an anionic denaturing agent. Contains a surfactant. When the analysis sample is a protein or complex protein (eg lipoprotein, glycoprotein, etc.), it is advantageous to include an anionic surfactant. 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, and among these, sodium ions are easily used. Among the alkyl sulfates, dodecyl sulfate (sodium salt, potassium salt, lithium salt, etc.) is preferred, and among them, sodium dodecyl sulfate (SDS) is most preferred. By incorporating SDS into the gel medium layer of the present invention, it becomes possible to efficiently separate proteins or complex proteins and measure their molecular weights. The content of the anionic surfactant as a denaturing agent is about 0.05w/v% to about 2.0w/v% of the gel forming solution.
v%, preferably about 0.1w/v% to about 1.5w/v
% range. 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. Further, a water-soluble polymer may be added to the polyacrylamide gel membrane depending on the case. As the water-soluble polymer, addition polymerization type or condensation polymerization type water-soluble polymer can be used. Specific examples of addition polymers include polyvinyl alcohol,
Examples include nonionic water-soluble polymers such as 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. The molecular weight of the water-soluble polymer is preferably in the range of about 10,000 to about 1,000,000. The water-soluble polymer is used in an amount ranging from about 2% to about 100%, and preferably from about 5% to about 50%, based on the total weight of monomer and crosslinker. The addition of a water-soluble polymer makes the polyacrylamide gel film plastic, which prevents it from breaking during cutting, and the gel film also has plasticity when drying, which has the advantage of improving brittleness and making it less likely to break. be. Furthermore, the viscosity of the gel film can be controlled by selecting the molecular weight and amount of the water-soluble polymer added. Preferably, the polyacrylamide gel membrane further contains agarose. Any agarose can be used, and any of low electroosmotic, medium electroosmotic, and high electroosmotic agaroses can be used. Examples of agarose that can be used include JP-A-55-5730
There are agaroses and the like disclosed in various publications such as Japanese Patent Application Laid-open No. 110946/1983 and Japanese Patent Application Publication No. 502098/1982. Agarose is used in a proportion of 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 monomer and crosslinking agent. It will be done. When the polyacrylamide gel film contains agarose, it is possible to control the viscosity of the gel forming solution to an appropriate level by changing the temperature of the gel forming solution. It has the advantage of being easier to mold in the molding operation. The gel medium layer can also contain a PH buffer. The buffer can be appropriately selected and used from known buffers that can buffer the pH value within the range of PH2.5 to 10.0, depending on the sample to be electrophoretically analyzed. Buffers that can be used are listed in "Chemical Handbook Basic Edition" edited by the Chemical Society of Japan (Tokyo, published by Maruzen Co., Ltd. in 1966) 1312
−1320 pages; “Latest electrophoresis methods” edited by Aoki and Nagai
(Tokyo, Hirokawa Shoten, published 1973, pages 320-322; "Data for Biochemical Research" (RM
Edited by C. Dawson et al., 2nd edition, Oxford at the
Clarendon Press, 1969) pages 476-508; "Biochemistry" 5 , 467 (1966),
“Analytical Biochemistry” 104, 300−310
(1980) and the like. Examples of buffers include barbital-containing buffers, tris(hydroxymethyl)aminomethane (Tris)-containing buffers, phosphate-containing buffers, borate-containing buffers, acetic acid or acetate-containing buffers. agents, buffers containing citric acid or citrate;
Buffers containing lactic acid or lactate, buffers containing glycine, N,N-bis(2-hydroxyethyl)
Glycine (Bicine), N-2-hydroxyethylpiprazine-N'-2-hydroxypropane-3
-Sulfonic acid (HEPPSO) or its salt, N-
2-Hydroxyethylpiperazine-N'-3-propanesulfonic acid (EPPS) or its salt, N-
Examples include [tris(hydroxymethyl)]-3-aminopropanesulfonic acid (TAPS) or a salt thereof, and acids, alkalis, or salts in combination with any of these as necessary. Specific examples of preferred buffers include potassium dihydrogen phosphate-disodium hydrogen phosphate, Tris/sodium borate, and Tris/sodium borate.
EDTA2Na salt, Tris/citric acid, sodium barbital/sodium acetate, sodium barbital/hydrochloric acid, barbital/sodium barbital, acetic acid/sodium acetate, lactic acid/sodium lactate, citric acid-disodium hydrogen phosphate, Bicine, HEPPSO, HEPPSO Sodium salt, EPPS, EPPS sodium salt, TAPS,
Examples include TAPS sodium salt. The electrophoretic medium layer (polyacrylamide gel membrane) in the electrophoretic medium material of the present invention is made of a monomer typified by acrylamide, a bifunctional allyl compound, or an acrylic compound (crosslinking agent) as described above. ), a water-soluble polymer, and agarose, are obtained by radical crosslinking polymerization of a monomer and a crosslinking agent in a substantially uniform aqueous solution, and three-dimensional crosslinking formed from the monomer and crosslinking agent. It is presumed that the polymer has a structure in which the water-soluble polymer and agarose are substantially dispersed, and the polymer chains of the latter two are entangled with the three-dimensional crosslinked 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,
"Electrophoresis" 1981, 2 , 213-219, same
1981, 2 , 220-228; Aoki and Nagai, eds., "Latest Electrophoresis Methods" (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-dimethylaminopropionitrile (DMAPN)/ammonium peroxodisulfate mixture, N,N,N',N'-tetramethylethylenediamine (TEMED)/ammonium peroxodisulfate mixture, TEMED. - Riboflavin mixture, TEMED, combination of riboflavin/hydrogen peroxide mixture and ultraviolet irradiation, etc. 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. The gel medium layer is formed by applying a gel-forming liquid by a known method onto the above-mentioned adhesive layer provided on a support having a smooth surface, and then cross-linking and polymerizing the gel-forming liquid to form a layered layer. It can be formed into. When cross-linking and polymerizing the gel-forming liquid on the surface of the support, a cover film,
It can be covered with a covering material such as a sheet or board. 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 μm or less, with a practically preferred range of about 4 μm to about 200 μm, and a particularly preferred range of about 4 μm to about 100 μm. 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 polyol compound is approximately
It is selected from the range of 5w/v% to about 40w/v%.
Among these, glycerin is particularly preferred. By incorporating a wetting agent, it is possible to prevent the gel film from drying out due to extreme water evaporation during storage, and it also prevents the occurrence of brittleness caused by extreme dryness and improves the physical properties of the gel film, such as preventing cracking. It has the advantage of being improved. 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 is possible to omit the complicated operation steps conventionally required in the electrophoresis operation of proteins or complex proteins. It becomes possible to carry out electrophoresis and staining operations with the medium layer (polyacrylamide gel membrane) still placed. Next, examples of the present invention will be shown. Example 1 Thickness with surface made hydrophilic by ultraviolet irradiation treatment
180μm polyethylene terephthalate (PET)
Approximately 0.5 μm thickness (solid content) on the sheet (support)
Apply a coating solution obtained by dissolving the cellulose derivatives listed in Table 1 in acetone so that the temperature is approximately 110°C.
was dried to form a cellulose derivative adhesive layer.

[Table] First, the adhesiveness between the PET sheet (support) and the adhesive layer was evaluated by the cross-cut method. As a result, Samples 1 to 3 (samples according to the present invention) had good adhesive properties. On top of each adhesive layer provided on the support, 9.5 g of acrylamide, 0.5 g of BIS, 3.58 g of disodium hydrogen phosphate dodechydrogen, 0.33 g of sodium dihydrogen phosphate dihydrate, and 0.10 g of SDS. contains
Ammonium peroxodisulfate (5% by weight) 1.3ml as a polymerization initiator in 100ml solution, TEMED33μ
The mixture was molded to a thickness of 0.5 mm to form a polyacrylamide gel film. In addition, a comparative sample was prepared by forming a polyacrylamide gel film on a PET sheet in the same manner except that no separate cellulose derivative adhesive layer 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, samples 1 to 3
(Sample according to the present invention) as a mounting layer had excellent mounting properties, but comparative samples had poor adhesion properties. Example 2 A PET sheet on which the same cellulose derivative adhesive layer as in Table 1 of Example 1 was formed was prepared, and 9.5 g of acrylamide, 0.5 g of BIS, and agarose 1600 (manufactured by Wako Pure Chemical Industries, Ltd.) were placed on this adhesive layer. ) 0.3 g, polyacrylamide 2.5 g, disodium hydrogen phosphate dodecahydrate 3.58 g, sodium dihydrogen phosphate dihydrate 0.33
ammonium persulfate (5% by weight) as a polymerization initiator to 100 ml of a solution consisting of
Three types of samples (Samples 1 to 3) were obtained by molding 1.3 ml of TEMED to a thickness of 0.5 mm to form a polyacrylamide gel film. A comparative sample was also prepared by directly forming the above polyacrylamide gel film on PET. This gel membrane was used for electrophoresis of standard proteins. The gel film was then coated with 0.1% Coomassie Blue R-250 (Color
Index Constitution Number 42660)
Staining was performed, and the state of adhesion between the support and the gel film during this dyeing process was observed. In a comparative test, the gel film was completely peeled off from the support as soon as it was immersed in the staining solution. On the other hand, in samples 1 to 3 (according to the present invention), the gel films were completely adhered to the support throughout the dyeing process. Furthermore, 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 cellulose derivative adhesive layer as in Table 1 of Example 1 was formed, and three types of samples (Samples 1 to 3) were prepared. Prepared. A comparative sample 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 while some peeling of the gel film was observed for the comparative samples, for samples 1 to 3 (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 Acrylamide was placed on a PET sheet on which the same cellulose derivative adhesive layer as in Table 1 of Example 1 was formed.
4.56g, BISO0.24g, polyacrylamide 1.2g,
Agarose 0.3g, Tris (hydroxymethyl)
Aminomethane [CAS Registry No.77-86-1]
1.08g, boric acid 0.55g and EDTA・2Na salt 93
ammonium persulfate (5wt%) as a polymerization initiator in a 100ml solution containing 20g of glycerin.
ml and TEMED33μ was applied to a thickness of 1 mm and polymerized in a nitrogen atmosphere to form a polyacrylamide film. E. coli plasmid pBR-322 with restriction enzyme AsuI
When the DNA degradation pattern was examined by ethidium staining, a normal staining pattern was found. The gel membrane was cut using a cutter knife to form a sample injection port, and the cut could be made sharply. Furthermore, it was found that the separated DNA hands could be fractionated with high precision.

Claims (1)

[Scope of Claims] 1. An electrophoretic medium material comprising a three-layer structure in which the following layers are sequentially laminated: [] a plastic support layer; [] an adhesive layer containing a cellulose derivative; and [] an acrylamide-based material. A polyacrylamide-based aqueous gel electrophoresis medium layer, which is formed by cross-linking polymerization of a compound and a cross-linking agent in the presence of water, and contains an anionic surfactant as a modifier. 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, wherein the anionic surfactant is an alkyl sulfate. 4. The electrophoretic medium material according to claim 1, wherein the anionic surfactant is sodium dodecyl sulfate. 5. The electrophoretic medium material according to claim 1, wherein the plastic support layer is made of polyethylene terephthalate. 6. The electrophoretic medium material according to claim 1, wherein the adhesive layer containing a cellulose derivative is a layer consisting essentially only of a cellulose derivative. 7. The electrophoretic medium material according to claim 1 or 6, wherein the cellulose derivative is selected from the group consisting of diacetylcellulose, triacetylcellulose, and nitrocellulose.