JPS63313056A - Production of gradient gel film for electrophoresis - Google Patents

Abstract

PURPOSE:To enhance productivity, reproducibility, etc., by maintaining the temp. at the time of mixing and compounding two kinds of monomer liquids and a polymn. reaction initiator liquid at the temp. higher than the temp. at which the components in a medium liquid precipitate and the temp. at which a gel-like material is generated. CONSTITUTION:The medium liquid 12 for electrophoretic sepn. is compounded by stirring and mixing the low- and high-concn. monomer liquids 1, 2 and the polymn. reaction initiator liquid 3 in a static mixer 7. Flow rate gradient liquid feed pumps 4, 5 are controlled by a controller 9a which is commanded by a personal computer 11, by which the monomer liquids are fed to the mixer 7. On the other hand, the liquid 3 is controlled by a controller 9b and is fed at a fixed rate to the mixer 7 by a constant flow rate liquid feed pump 6. These liquids are stirred and mixed in the mixer 7, by which the medium liquid 12 is prepd. This liquid 12 is supplied to a coating head 8 and is coated on a web 14. Cooling liquid is passed by a cold retaining water inlet 25 to a cold retaining water outlet 26 to maintain the temp. in the mixer 7 at the temp. higher than the temp. at which the components in the medium liquid precipitate and lower than the temp. at which the gel-like material is generated. The mixing and stirring are executed at said temp.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing a gradient gel membrane for electrophoresis for separating and analyzing proteins, particularly proteins with a wide molecular weight distribution range such as urinary proteins. In particular, it relates to the preparation of a medium solution for electrophoretic separation.

[Prior Art] Conventionally, in plate electrophoresis, acrylamide polymer concentration gradient gels that do not have self-supporting properties require gels with different concentrations to be deposited on one support or between two supports. It has been used as a membrane by forming a layer in the direction of electrophoresis.

However, the method of forming and using a gel on a support is
When forming a gel on a support, when setting it in an electrophoresis tank,
While storing the gel or adding an analytical sample, it is possible to accidentally break the gel or drop something other than the sample onto the gel, damaging the gel. was necessary.

On the other hand, in the vertical electrophoresis method, a mold is made from two glass plates, etc., a gel is formed in the mold, and the mold is held vertically for electrophoretic analysis.In the vertical electrophoresis method, the thickness of the mold is made uniform. It is difficult to do this, and requires a high level of skill in operation, as the gel forming solution must be injected into a narrow mold before it gels.

Furthermore, since a glass plate is used, there is a drawback that the glass plate is easily damaged.

In recent years, as a method for industrially producing gradient gel materials for electrophoresis, an aqueous solution or aqueous dispersion of a mixture of an acrylamide monomer and a crosslinking agent is prepared, and a free radical that absorbs light irradiation and initiates the polymerization of the 7-mer After adding the generated material, the solution is formed into the desired gel product, and the formed solution is irradiated with light and then the 7-mer solution or dispersion is polymerized and crosslinked to change the porosity of the gel. A method for producing an electrophoretic gradient gel including a step of adjusting the light irradiation time applied to the monomer solution (Japanese Patent Application Laid-open No. 61-2
(See Publication No. 8512).

Two types of aqueous solutions containing Zaranimata monomers, crosslinking agents, and polymerization initiators and having different concentrations are introduced into a molding machine while being mixed while gradually changing the mixing ratio. A method for producing a gradient gel for electrophoresis having a polymer concentration gradient in the direction of electrophoresis by completing the polymerization of a polymer and a crosslinking agent (Japanese Patent Publication No. 61-22903, Japanese Patent Publication No. 61-3
9817) etc. are disclosed.

Furthermore, the medium liquid for electrophoretic separation is prepared by adding a certain amount of polymerization reaction initiator liquid to two types of monomer liquid, concentrated and light, with the flow rate ratio continuously changed, and the liquid is applied onto the web. method was used.

[Problems to be solved by the invention] However, the first manufacturing method has high costs due to poor light irradiation equipment and poor productivity, poor resolution due to the thick gel, and problems with the reaction initiator. Even after completion, polymerization and crosslinking reactions occur in the presence of light, resulting in problems such as the inability to obtain stable and reproducible gradient gels.The next production method is still a batch production method. Productivity is poor due to
Further, when the electrophoretic separation medium liquid is branched into the molding device, it is difficult to branch it uniformly, and there are problems such as the inability to obtain a reproducible gradient gel. Furthermore, in the method of mixing a fixed amount of polymerization reaction initiator liquid with two types of concentrated and light acrylamide monomer liquids using a mixing device, the flow rate ratio of which is continuously changed, an unstable state occurs in which foreign matter is generated in the medium liquid. It had some problems.

An object of the present invention is to solve the above-mentioned problems and provide a method for producing a gradient gel membrane for protein analysis electrophoresis, which has high productivity, high reproducibility, and good stability.

[Means for Solving the Problems] As a result of intensive studies on the above-mentioned problems, the inventors of the present invention found that the foreign matter in the medium liquid is a precipitate or gel-like substance of components generated due to the temperature of the mixing/stirring device. found.

The object of the present invention is to provide a method for producing a gradient gel membrane for electrophoresis in which a medium liquid for electrophoretic separation is applied onto a continuously running web, in which the medium liquid for electrophoretic separation is
When mixing the monomer liquid of two types, concentrated and light, and the polymerization reaction initiator liquid with the flow rate ratio continuously changed using a stirring device, the temperature is set higher than the temperature at which the components in the medium liquid precipitate, resulting in a gel-like substance. This is achieved by a method for producing a gradient gel membrane for electrophoresis that does not contain urea as a denaturing agent, which is carried out by maintaining the temperature within a temperature lower than the temperature at which .

The running web in the present invention may be made of any material as long as it is in the form of a sheet with good flatness, is non-conductive, and is substantially water-impermeable, such as polyethylene terephthalate or bisphenol A polycarbonate. Polyesters such as polymethyl methacrylate, polyethylene, polystyrene, vinyl polymers such as polyvinyl chloride, polyamides such as nylon, and copolymers thereof (eg, vinylidene chloride/vinyl chloride copolymer) are preferably used.

The medium liquid for electrophoretic separation in the present invention may be anything as long as it can form a medium membrane for electrophoretic separation, and typical examples include acrylamide gel, agarose gel, starch gel, agar gel, and cellulose acetate. A liquid that is used as a raw material for porous membranes, filter paper, etc.

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. A surfactant can be included.

When the analysis sample is a protein or complex protein (eg, riboprotein, glycoprotein, etc.), it is often preferable or necessary to include an anionic surfactant.

Of course, the anionic surfactant may not be included in the gel medium layer.

For example, a gel medium layer that does not contain an anionic surfactant can be used for purposes such as genetic disease diagnosis based on DNA fragment analysis or DNA structure analysis using restriction enzyme digestion.

By including an anionic surfactant in the electrophoresis medium layer, efficient separation of proteins or complex proteins and determination of their molecular fn1lll becomes possible.

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. S
By incorporating DS into the gel medium layer of the present invention, it becomes possible to efficiently separate proteins or complex proteins and determine their molecular weights apj.

The content of the anionic surfactant as a denaturing agent is about 0.05 v/v% to about 2.0% based on the gel forming solution. Qw/v%
, preferably about 0. It ranges from h/v% to about 1.5w/v%.

In the present invention, methods for applying the medium liquid for electrophoretic separation include slide bead coating, extrusion coating,
Application heads such as hopper coat and curtain coat are used. The thickness of the media membrane to be coated is selected depending on the purpose of separation, but is usually from 50 μm to about 2.0+n, preferably from about 100 μm to about 1.01%, preferably from about 70 μm to about 1.0% in the case of a porous membrane or filter paper. This is considered to be within the scope of this matter.

The gradient gel membrane for electrophoresis in the present invention refers to one having a concentration gradient within one product unit length.

Although any type of stirring device may be used in the present invention, a static mixer is most suitable.

A static mixer is a mixer that stirs the fluid by its own movement when the fluid is passed through the mixer. Examples of tubular types include a spiral tube, an alternating left and right spiral, or a propeller with baffles. be.

Embodiments of the present invention will be described using figures.

FIG. 1 is a process diagram of one embodiment of the method for producing a gradient gel membrane for electrophoresis according to the present invention.

The electrophoresis medium liquid 12 is prepared by stirring and mixing a low concentration monomer liquid 1, a high concentration monomer liquid 2, and a polymerization reaction initiator liquid 3 in a static mixer 7.

FIG. 3(a) is an explanatory diagram of the relationship between the flow rate ratios of the two types of concentrated and light seven-day liquids of the present invention. Continuously changing the flow rate means that, for example, initially high concentration monomer liquid 2 is Oml/
m 1 n s low concentration monomer liquid 1 is 38 m110+
It starts with a flow rate ratio of in, but if the product unit length of the gradient gel membrane for electrophoresis is g, the flow rate is shown as the solid line for high concentration monomer liquid 2 and the dotted line for low concentration monomer liquid 1 in the figure. The total flow rate is constant at 38 ml/l lin, but the concentration changes continuously. The end of the product unit length g is 38ml for high concentration monomer liquid 2.
/sin, the low concentration monomer liquid 1 becomes Oml/a+in and becomes a highly concentrated membrane component. Then, the next product unit length goes from high concentration to low concentration. In order to repeatedly change the flow rate ratio as described above, in FIG. be done.

On the other hand, the polymerization reaction initiator liquid 3 is controlled by the controller 9b by the constant flow liquid sending pump 6, and a constant amount is sent to the static mixer 7, where it is stirred and mixed to form the electrophoretic separation medium liquid 12. The electrophoretic separation medium liquid 12 is supplied to the coating head 8 and is coated onto the web 14 .

At this time, as shown in FIG. 2, a jacket 24 of the static mixer is provided as a jacket 24 as an outer jacket of the housing 22 which seals the element 21 of the static mixer.
4, cooling water is flowed from the cold water inlet 25 to the cold water outlet 2B, and the temperature inside the static mixer is kept within a temperature higher than the temperature at which the components in the medium liquid precipitate and lower than the temperature at which gel-like substances are generated.・Stir.

FIG. 3(b) is a plan view of a gradient gel membrane for electrophoresis made using the concentration ratio shown in FIG. 3(a), and FIG.
) is a side view of FIG. 3(b).

The gap t between the product unit lengths 9 in FIG. 2 is punched out as a sample injection port, and is used as the sample injection port with a cover sheet or the like.

[Function] The present invention provides a method for producing a gradient gel membrane for electrophoresis in which a medium liquid for electrophoretic separation is applied onto a continuously running web, in which the medium liquid for electrophoretic separation is continuously applied at a flow rate ratio of When mixing the two types of monomer liquids (concentrated and light) and the polymerization reaction initiator liquid with a stirring/mixing device, the temperature is higher than the temperature at which the components in the medium liquid precipitate, and a gel-like substance is generated. Stable production is made possible by maintaining the temperature at a temperature lower than the temperature at which the gel is produced, for example, in the case of acrylamide gel, at 10 to 15°C. That is, when the temperature is below 10°C, precipitation of components in the medium occurs, and when the temperature is above 15°C, a gel-like substance is generated due to polymerization within the static mixer. By doing so, the medium liquid for electrophoretic separation is sent to the coating head without generating foreign matter, so it is possible to stably produce a coating film with a clear density gradient with high reproducibility and good stability of the medium liquid. You will be able to do it.

[Example] An example of the present invention will be described using FIGS. 1 and 2.

As medium solution for electrophoretic separation, low concentration monomer solution 1, aryl amide Old... Old... 51
8 fN, N'-methylenebisacrylamide
・・・ 27g Sodium dodecyl sulfate ・・・・・・
...... 9g [pH buffer] 1.5 M -Trls-HCl (pH 8,9) ++
+++++++ 2250ml deionized water up to
Create 9000m1.

Arilylamide as high concentration monomer solution 2
Old... 1000g N, N'-methylenebisacrylamide... 90g Sodium dodecyl sulfate... Old... 9g [p11 buffer] 1.5 M -Trls-hydrochloric acid (p) 18. 9) +
++・++++ 2250ml deionized water up t
o Make 9G00ml.

Note Tr1s Nidris(hydroxymethyl)aminomethane Next, as polymerization reaction initiator solution 3, 2.5% ammonium beroxonisulfate aqueous solution...
218 ml 25% N, N, N', N'-tetramethylethylenediamine solution 2.25 mlO,3
75% Riboflavin Phosphate Sodium Salt Aqueous Solution Make 150 ml of a mixed solution and put each of the three solutions into a tank.

Using the flow rate gradient liquid sending pumps 4 and 5, the sum of the flow rates of the low concentration upper Zimmer liquid 1 and the high concentration monomer liquid 2 was set to 38 m1/i+in, and the flow rate of the polymerization reaction initiator liquid 3 was set to 1.55 m1/akin.
is fed into the static mixer 7 using the metering liquid feed pump 6.

Inside the static mixer 7, a jacketed cooling device as shown in Figure 2 is used to keep the temperature inside the static mixer 1.
It was kept at 3°C. The above three liquids are mixed and stirred by a self-scissor blade, and an electrophoretic separation medium liquid 12 is completed.

The electrophoretic separation medium liquid 12 is applied to the web 1 from the coating head 8.
4.

At that time, the flow rate ratio between the low concentration monomer liquid 1 and the high concentration monomer liquid 2 is as shown in Figure 3 (a), and the concentration of the applied gel film is as shown in Figure 3 (b) and Figure 3. The state will be as shown in (c).

In addition, the product as a medium membrane for electrophoretic separation is prepared by a process of pasting spacers on both ends of the web in advance and a curing treatment after application, as disclosed in, for example, Japanese Patent Application Laid-open No. 1311147. process, the process of punching out a comb as a sample injection port, the process of pasting a cover sheet,
It becomes a product only after going through processes such as cutting it to the product length.

However, these steps have no relation to the present invention no matter what method or means is used.

[Effects of the Invention] The present invention provides a method for producing a gradient gel membrane for electrophoresis in which a medium liquid for electrophoretic separation is continuously applied onto a continuously running web. The temperature at which the two types of monomer liquid and polymerization reaction initiator liquid with different flow rate ratios are mixed using a stirring/mixing device is set to be higher than the temperature at which the components in the medium liquid precipitate. The method of manufacturing gradient membranes for electrophoresis, which is characterized by maintaining the temperature within a temperature lower than the temperature at which substances are generated, has higher productivity and reproducibility than conventional methods.
We were able to manufacture a gradient gel membrane for electrophoresis with good stability of the medium solution.

[Brief explanation of the drawing]

Figure 1 is a process diagram of the method for producing a gradient gel membrane for electrophoresis according to the present invention, and Figure 2 is a diagram showing the temperature inside the stirring/mixing device according to the present invention to prevent components in the medium from precipitating and gel-like substances. This is an example of a device that maintains the temperature within L5 at which the temperature does not occur. Figure 3 is an explanatory diagram (a) of the relationship between the flow rate ratios of the two types of concentrated and light monomer solutions of the present invention, and a plan view of the gradient gel membrane for electrophoresis of the present invention made with the concentration ratios shown in (a). b) and a cross-sectional view (c). 1...Low concentration monomer liquid 2...High concentration monomer liquid 3...Polymerization reaction initiator liquid 4.5...Flow rate gradient liquid feeding pump 6...Constant flow liquid feeding pump 7...Stirring・Mixing device (static mixer 8...
- Coating heads 9a, 9b... Controller 10... Computer linkage adapter 11...
Personal computer 12... Electrophoretic separation medium liquid 13... Coating roller 14... Web 21... Element (static mixer) 22...
...Housing 23...Flange 24...Jacket 25...Insulated water inlet 26...Insulated water outlet fee Attorney Patent attorney (8107) Kiyoshi Sasaki (and 3 others) 2 Shimadanki t/7- Liquid 3: 1GJj4#L? 6siFIl decision 4.5・flow 17
Red Liao liquid positive 7゛61! Lingyo I liquid go Q7゛7 Static Mi 6000-(Shoya fJ 2 bi) Volume 1) 8
: To cool down, pull, 9b, pull the μ roller; (p, Fog procedure amendment 1. Display of the case 1985 Patent Application No. 147832 2, Title of the invention Gradient glue membrane for electrophoresis Process for manufacturing 3. Relationship with the person making the amendment: Patent applicant name: (520) Fuji Photo Film Co., Ltd. 4, Agent 7. Subject of amendment: "Detailed description of the invention" column 8 of the specification. , Contents of amendment: "Detailed description of the invention" in the specification
The following column (1) “,” on page 3, line 11 of the specification.
” to be deleted. (2) After “Reference” on page 3, line 15 of the same disease, “
Insert "is disclosed." (3) “Polyamide, etc.” on page 6, line 10 of the same book
is corrected to "polyamide". (4) "Agar gel," on page 6, line 16 of the same book, was replaced with "
Correct with "agar gel". (5) Delete "cellulose acetate porous membrane, filter paper" on page 6, line 17 of the same book. (6) "Ionicity" on page 7, line 3 and line 17 of the same book is corrected to "ion". (7) In the same book, page 9, lines 1 and 2, [, in the case of porous membranes and filter paper, delete from about 70- to about 1.0 mm J. (8) In the same book, page 10, line 18, "to control" is amended to "to be controlled." (9) "Test amount" in line 8 of page 15 of the same book is "sample"
to correct.

Claims (1)

[Claims] In a method for producing a gradient gel membrane for electrophoresis in which a medium liquid for electrophoretic separation is applied onto a continuously running web, the medium liquid for electrophoretic separation is divided into concentrated and light liquids in which the flow rate ratio is continuously changed. The temperature when mixing the seed monomer liquid and the polymerization reaction initiator liquid with a stirring/mixing device is kept within a temperature higher than the temperature at which the components in the medium liquid precipitate and lower than the temperature at which a gel-like substance is generated. A method for producing a gradient gel membrane for electrophoresis that does not contain urea as a denaturing agent.