JPH065226B2 - Method for producing gradient gel membrane for electrophoresis - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a gradient gel membrane for electrophoresis for separating and analyzing a protein, particularly a protein having a wide molecular weight distribution range such as a protein in urine. Is.

[Conventional technology]

Conventionally, in flat-plate electrophoresis, a high-concentration acrylamide gel with no self-supporting property is formed by layering gels with different concentrations on one support or between two supports in the electrophoresis direction. Formed and used as a film.

However, the method of forming a gel on a support and using it is
When forming a gel on the support, when setting in the electrophoresis tank,
During storage of the gel or when adding an analytical sample, the gel may be broken by mistake, or something other than the sample may be dropped on the gel to damage the gel. Was necessary.

On the other hand, in a vertical electrophoresis method in which a mold is made with two glass plates, a gel is formed in the mold, and electrophoretic analysis is performed while the mold is held vertically, the mold thickness is made uniform. However, it requires a high degree of skill in operation such that the gel solution must be injected into a narrow mold before the gel-forming solution gels.

In addition, since the glass plate is used, there is a drawback that it is easily damaged.

In recent years, as a method for industrially producing a gradient gel substance for electrophoresis, an aqueous solution or an aqueous dispersion of a mixture of an acrylamide monomer and a cross-linking agent is prepared, and a free radical generating substance that absorbs light irradiation and starts polymerization of the monomer is prepared. After addition, the solution is formed into the desired gel product form,
A method for producing an electrophoretic gradient gel including a step of adjusting the light irradiation time applied to the monomer solution in order to change the porosity of the gel when the formed solution is subjected to crosslinking by polymerizing the monomer solution or the dispersion by irradiating the solution ( JP-A-61-2
Japanese Patent No. 8512) is disclosed. Furthermore, two kinds of aqueous solutions containing a monomer, a cross-linking agent and a polymerization initiator and having different concentrations are introduced into a molding machine while mixing while gradually changing the mixing ratio, and a single amount is charged in the molding machine. A method for producing a gradient gel for electrophoresis having a concentration gradient of the polymer in the electrophoretic direction by completing the polymerization between the body and the cross-linking agent (see JP-B-61-22903, JP-B-61-39617) and the like. It is disclosed.

However, the former method is costly due to light irradiation equipment and poor productivity, and the resolution is poor due to the thick and thick gel, and the reaction initiator polymerizes in the presence of light even after the production is completed.
There is a problem that a gradient gel with stability and reproducibility cannot be obtained due to a crosslinking reaction. The latter is also a batch type manufacturing method, so productivity is poor, and a medium solution for electrophoretic separation is molded. It has a problem that it is difficult to uniformly branch when flowing into the vessel and a reproducible gradient gel cannot be obtained.

As a method for producing a gradient gel film for electrophoresis, which solves the above problems and has high productivity and high reproducibility, the applicant of the present invention has previously used two types of concentrated and light monomer liquids in which the flow ratio was continuously changed. An electrophoretic gradient gel in which a certain amount of a polymerization reaction initiator liquid is mixed with a static mixer to prepare an electrophoretic separation medium liquid, and the electrophoretic separation medium liquid is applied onto a continuously running web. A method of manufacturing the membrane was proposed.

[Problems to be solved by the invention]

The coating method is an excellent method and is suitable for production as a high-grade electrophoretic gradient film because of the switching of the liquid volume ratio of the dark and light two liquids and the multi-stage concentration gradient. A method for producing a reproducible and highly productive gradient gel membrane for electrophoresis having a concentration gradient of several steps has been desired.

An object of the present invention is to address the above problems and to provide a method for producing a gradient membrane for protein separation analysis electrophoresis having a simple concentration gradient of several steps with high reproducibility and high productivity.

[Means for solving problems]

The above-mentioned object of the present invention is a method for producing a gradient gel membrane for protein analysis electrophoresis in which a medium solution for electrophoretic separation is applied onto a continuously running web, and a monomer solution having different concentrations at a plurality of stages and respective polymerization initiators. Mix and prepare the liquid and the liquid, respectively, and supply the mixed liquids to the plurality of liquid supply ports of the coating head in the order of concentration and separately, and spread the liquid in each slot width to horizontally mix multiple liquids. Characterized in that it is applied on the web from the slot opening as a single-row coating film,
This is achieved by a method for producing a gradient gel membrane for electrophoresis containing an anionic surfactant as a denaturant.

In the present invention, the continuously running web may be made of any material as long as it is a sheet having good flatness, non-conductive and substantially impermeable to water, such as polyethylene terephthalate and bisphenol. Polyesters such as A's polycarbonate, polymethylmethacrylate,
Vinyl-based polymers such as polyethylene, polystyrene and polyvinyl chloride, polyamides such as nylon, and copolymers thereof (eg, vinylidene chloride / vinyl chloride copolymer) are preferably used.

The medium solution for electrophoretic separation in the present invention may be anything as long as it can form a medium film for electrophoretic separation, and typical examples thereof include a raw material solution such as acrylamide gel, agarose gel, starch gel, agar gel and the like. It means the liquid that becomes.

The electrophoretic medium material of the present invention is advantageously used mainly for the analysis of proteins or complex proteins (for example, lipoproteins, glycoproteins, etc.), and the electrophoretic medium layer contains an anionic interface as a denaturant. An activator can be included. In addition to this, when a gradient membrane for electrophoresis is used for the purpose of determining the nucleotide sequence of DNA, urea is used as a denaturing agent. Since the isoelectric point is polyvalent and the charge is not constant when urea is used, it is effective to use an anionic surfactant that gives a constant charge per molecule. Thus, when the analysis sample is a protein or complex protein (eg lipoprotein, glycoprotein, etc.), it is often preferable or essential to include an anionic surfactant. Of course, the anionic surfactant may not be contained in the gel medium layer. For example, a gel medium layer containing no anionic surfactant is
It can be used for the purpose of genetic disease diagnosis based on DNA fragment analysis or DNA structure analysis utilizing restriction enzyme decomposition.

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

Examples of the anionic surfactant include alkylsulfates, and alkylsulfates having a long-chain alkyl group having 10 or more carbon atoms are particularly preferably used. Alkali metal ions such as sodium ions, potassium ions and lithium ions are generally used as the cations that form salts, and of these, sodium ions are easy to use. Among the alkyl sulfates, dodecyl sulfate (sodium salt, calcium salt, lithium salt, etc.) is preferable,
Of these, sodium dodecyl sulfate (SDS) is most preferable. The inclusion of SDS in the gel medium layer of the present invention enables efficient separation of proteins or complex proteins and measurement of their molecular weight.

The content of the anionic surfactant as a modifier is about 0.05 W / V% to about 2.0 W / V%, preferably about 0.1 W / V% to about 1.5 W / V with respect to the gel forming solution. % Range.

As a method for applying the medium liquid for electrophoretic separation in the present invention, slide bead coating, extrusion coating, hopper coating, curtain coating and the like are used.
The thickness of the medium liquid to be applied is selected depending on the purpose of separation, but is usually in the range of 50 μm to about 2.0 mm, preferably about 100 μm to about 1.0 mm.

The gradient gel film for electrophoresis in the present invention refers to one having a stepwise concentration gradient within one product unit length. In the present invention, the term "monomer liquid having different concentrations in multiple stages" refers to a liquid in which the concentration is changed in multiple stages within 3 to 35 W / V% depending on the purpose of use.

A mixing device for mixing each concentration of the monomer liquid and each of the polymerization initiator liquids may be tank mixing or any other method, but a preferred method is a static mixer. A static mixer is a mixer that agitates by the motion of the fluid itself when the fluid is passed through the mixer, and as a tubular one, a spiral tube, alternating left and right spirals, or a propeller with baffles, etc. There is.

Embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a process chart of one embodiment of the method for producing a gradient gel film for electrophoresis of the present invention. In this embodiment, an example in which the liquid concentration is divided into 5 stages will be described.

The electrophoretic separation medium liquid 6 (a to e) is the monomer liquid 1
(A to e), the polymerization reaction initiator liquid 2 (a to e) corresponding to the monomer liquid is adjusted in each tank, and each is adjusted by the constant flow rate pumps 3 (a to e) and 4 (a to e). Then, the mixture medium 6 for electrophoresis, which is each mixed solution, is sent by pressure and is stirred and mixed in the static mixer 5 (a to e).
Prepare (a to e).

The five types of liquids 6 (a to e) are pumps 3 (a to e) 4 (a
~ E) pressure applied to each of the liquid supply ports 14 of the coating head 7
(A) to (e), and are applied side by side on the web 9 on the coating roller 8 through the slot opening 16. A differential pressure chamber 10 is provided to apply back pressure to the rear side of the coating liquid bead for stability of coating.

FIG. 2 is a plan view (a), a side view (b), and a cross-sectional view (c) illustrating a state in which five kinds of electrophoretic separation medium liquids 6 (a to e) having different concentrations are applied on the web 9. ).

The mixed liquids 6 (a to e) are respectively supplied to the supply ports 14 (a to e) of the coating head 7 and are applied on the web 9 through the slot openings 16 as a horizontal example. That is, as the gradient gel film for electrophoresis, product units are formed in the width direction of the web, and the web is processed by a cutting machine.

FIG. 3 is a side sectional view of the coating head used in the present invention,
The figure shows a front sectional view of FIG.

In FIG. 3 and FIG. 4, the five kinds of liquids are pump 17 respectively.
(A to e), the liquid supply port 14 (a to
Liquid is sent to e). The respective liquids are spread in the width direction in the coating head 7 by the slots 15 (a to e) without a gear bit. In this case, the width of each slot is narrow and the spread angle of the liquid in the slot can be set to θ ≦ 40 °, so that the liquid spreads naturally and the accumulation of liquid and the generation of foreign matter caused by it do not occur. The adjacent liquids are in contact with each other in front of the slot opening 16, and the liquids form a coating film in a horizontal row in the slot opening 16 and are applied onto the web 9.

[Work]

In a method for producing a gradient gel membrane for protein analysis electrophoresis, in which a medium solution for electrophoretic separation is applied on a continuously running web, a monomer solution having different concentrations in a plurality of stages and respective polymerization initiator solutions are mixed and mixed. Liquid is prepared, and each of the mixed liquids is arranged separately in the order of concentration to a plurality of liquid supply ports of the coating head, and the liquids are spread over each slot width, and a plurality of mixed liquids form a straight line coating film from the slot opening. A product unit length is formed in the web width direction as the electrophoresis gradient film by the method for producing a gradient gel film for protein analysis electrophoresis, which is characterized by being applied onto a web. Since the mixed solution of each step concentration is prepared under constant conditions without any change in the concentration in the process, the quality is stable, and in the coating head, the coating solution (mixed solution) naturally spreads to form a coating film. Since it is applied, a good quality coating film can be obtained without the generation of foreign matter due to stagnation, and the quality and reproducibility of the product are good and the productivity is high due to stable manufacturing conditions.

〔Example〕

The present invention will be described with reference to one embodiment with reference to FIGS.

Medium liquid for electrophoretic separation As low-concentration monomer liquid 1a Acrylamide 102.6 g N, N'-methylenebisacrylamide 5.4 g Sodium dodecyl sulfate 1.8 g [pH buffer] 1.5 M-Tris-hydrochloric acid (pH 8.9) Add 450ml deionized water to make 1800ml.

High-concentration monomer liquid 1e Acrylamide 342g N, N'-methylenebisacrylamide 18g Sodium dodecyl sulfate 1.8g [pH buffer] 1.5M-Tris-hydrochloric acid (pH 8.9) 450ml Deionized water is added Bring to 1800 ml. Further, as a liquid having a concentration between them, a low-medium concentration monomer liquid 1b, a medium-concentration monomer liquid 1c, and a high-medium concentration monomer liquid 1d are prepared.
Each of the monomer liquids 1b, 1c and 1d is the same as 1a and 1e except that the respective amounts of acrylamide and N, N'-methylenebisacrylamide are equally spaced with a value between 1a and 1e.

(Note) Tris: Tris (hydroxymethyl) aminomethane Next, 2.5% ammonium peroxodisulfate aqueous solution as polymerization reaction initiator liquid 2 (a to e) ... 43.2 ml 25% N, N, N ', N'-tetramethylethylenediamine liquid ... 0.45 ml 0.375% riboflavin phosphate sodium salt aqueous solution ... Make a mixed solution of 30 ml, and then use the constant flow rate feed pumps 3 (a to e) to remove each monomer solution 1
(A to e) is 7.6 ml / min, and in addition, polymerization reaction initiator liquid 2 (a to e) is added by a constant flow rate liquid feed pump 4 to a flow rate of 0.311 ml.
/ Min each, and liquid is fed into the Stykix mixer 5 (a to e) together.

In the static mixer 5 (a-e), the above-mentioned two liquids are mixed and stirred by their own force to form a mixed liquid for electrophoretic separation 6 (a-e).

The electrophoretic separation medium liquid 6 (a to e), which is a mixed liquid, is supplied from the liquid supply ports 14 (a to e) of the coating head 7, and is applied onto the web 9 through the slot openings 16.

The state of application of the electrophoretic separation medium liquids 6 (a to e) at each concentration at that time is as shown in FIG. 2 (a), and the concentration of the applied gel film is divided into 5 stages and is continuously. Is ready to be applied to.

In addition, the product as a medium film for electrophoretic separation is a process of curing after coating, a process of punching a comb (comb) as a sample injection port, a process of applying a cover sheet, a process of cutting to a product length, etc. It becomes a product only after going through.

However, these methods are not related to the present invention by using any method or means.

〔The invention's effect〕

The present invention is a method for producing a gradient gel membrane for protein analysis electrophoresis in which a medium solution for electrophoretic separation is applied on a continuously running web, and a monomer solution having different concentrations in a plurality of stages and respective polymerization reaction initiator solutions Are mixed and prepared, and the mixed liquids are lined up in multiple supply ports of the coating head in the order of concentration and supplied separately, and the liquids are spread in each slot width, and multiple types of mixed liquids are applied in a horizontal row. A gradient gel membrane for electrophoresis having a simple concentration gradient of several steps by a method for producing a gradient gel membrane for electrophoresis containing an anionic surfactant as a denaturant characterized by being applied onto a web through a slot opening as a membrane Was manufactured with good productivity, stable product quality, and good reproducibility.

[Brief description of drawings]

FIG. 1 is a process diagram of a method for producing an electrophoretic gradient gel according to the present invention, and FIG. 2 is an explanatory diagram of a coating state of 5 kinds of medium liquids for electrophoresis separation of dark and light according to the present invention. FIG. 3 is a side sectional view of the coating head according to the present invention, and FIG. 4 is a front sectional view of FIG. 1 (a to e) ... Monomer liquid, 2 ... 2 (a to e) ... Polymerization reaction initiator liquid, 3 (a to e), 4 (ae) ... Constant flow rate liquid feed pump, 5 (a to e) ... Status Mixer, 6 (a-
e) ... Electrophoretic separation medium solution, 7 ... Coating head, 8 ... Coating roller, 9 ... Web, 10 ... Differential pressure chamber, 11 ... Controller, 12 ... Computer linkage cage adapter, 13 ... Web, 14 (a to e) … Filling port,
15 (a to e) ... Slot, 16 ... Slot opening, 17
(A to e) ... Metering pump.

Claims (2)

[Claims] 1. A method for producing a gradient gel membrane for protein analysis electrophoresis, wherein a medium solution for electrophoretic separation is applied onto a continuously running web, and a monomer solution having different concentrations in a plurality of stages and respective polymerization initiators. Mix and prepare liquids, and supply the mixed liquids to the multiple liquid supply ports of the coating bed separately in the order of concentration, and spread the liquid into each slot width. The method for producing a gradient gel film for electrophoresis containing an anionic surfactant as a modifier, characterized in that the coating film is applied onto the web through a slot opening. 2. The gradient gel membrane for protein analysis electrophoresis according to claim 1, wherein the change in concentration of the monomer liquids having different concentrations is 3 to 35 W / V%. Manufacturing method.