JPH043823B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to electrophoretic media materials. More specifically, the present invention relates to an electrophoresis medium material suitable for analysis of biological substances such as proteins and nucleic acids, and analysis using autoradiography.

[Background of the Invention] Electrophoretic analysis utilizes the fact that the mobility caused by an electric field in a conductive medium differs depending on the substance. In recent years, electrophoretic analysis has been widely used for the analysis of biological components, and in particular, it is frequently used for the purpose of analyzing biopolymers such as DNA and proteins in biochemical tests for disease diagnosis.

For details on the electrophoretic analysis method and the electrophoretic media membrane used in it, please refer to "Electrophoretic Experimental Methods (Revised 5th Edition)" edited by the Electrophoretic Society (Bunkodo, published in 1975).
Aoki and Nagai (eds.) “Latest Electrophoresis” (Hirokawa Shoten,
(published in 1973), etc. As is clear from the above literature, there are various types of electrophoretic analysis methods. Among these various electrophoretic analysis methods, the flat plate electrophoretic analysis method is particularly important.

BACKGROUND ART Plate electrophoresis analysis has become an indispensable means for separating and analyzing biological substances such as proteins and nucleic acids in the fields of biochemistry and medicine. In particular, the determination of DNA base sequences, which is important in genetic engineering and genetic disease research, requires mutual comparison of the migration distances of DNA fragments specifically degraded or synthesized for four types of bases. Therefore, it is essential to use a flat plate type electrophoretic analysis method.

In general, a plate-type electrophoretic analysis method consists of a sheet-like support and an electrophoresis medium membrane. In the conventional flat plate electrophoresis analysis method,
A glass plate is used as the support, and a gel membrane manufactured by coating or casting a film-forming material such as agar, cellulose, cellulose acetate, starch, silica gel, or polyacrylamide on the support is used as the electrophoresis medium membrane. ing. When analyzing a sample, the electrophoresis medium membrane mentioned above is impregnated with a buffer solution, the sample is attached onto it, a voltage is applied to both ends of the support, and the membrane is developed (moved) on or inside the support. ). Then, the sample on the support is dyed, and the optical density of the dyed sample is measured to perform quantitative or qualitative analysis of each component of the substance.

In addition, instead of the above-mentioned analysis by staining and optical density measurement, target components in the sample are labeled with radioisotopes, separated by electrophoresis, and then separated images of the target components in the sample are obtained using autoradiography. This is often done. In this method using autoradiography, an electrophoresis medium film containing a sample labeled with a radioisotope and a photographic film that records radioisotope radiation are stacked on top of each other and left in a dark place (this is called an exposure operation). )I do.

In conventional flat-plate electrophoresis analysis methods, when a non-self-supporting polymer gel such as agarose or acrylamide is used as the electrophoresis medium membrane, it is necessary to A method of forming a gel as a film-like material (layered material) in between has been used.

However, with the method of forming a gel film on a single support and using it for analysis as it is, mistakes can be made during storage of the gel film, when setting it in the electrophoresis tank, or when adding the analysis sample. The gel membrane could be damaged by breaking the gel membrane or by dropping something other than the sample onto the gel membrane, so careful handling and skill were required.

On the other hand, the method of forming a gel film between two supports and using it for analysis requires fewer operational precautions, but it may be difficult to make the gel thickness uniform. However, a high level of skill was required in terms of operation, as the gel forming solution had to be injected into a narrow mold before it gelled. In particular, in DNA base sequencing operations,
Although it is desirable to make long gels so that as many DNA fragments as possible can be analyzed in a single gel, such gels are difficult to manufacture and handle.

Furthermore, a method is also used in which a gel film is formed on a single support, a cover sheet is provided on the gel film, and storage and analysis operations are performed. However, even if this method is used, it is necessary to remove the cover sheet when performing an exposure operation in autoradiography. The reason why it is necessary to remove the cover sheet in autoradiographic analysis is to prevent radiation from the isotope of the sample in the gel film from being absorbed as it passes through the cover sheet and weakening its intensity. When removing this cover sheet, you may accidentally break the gel film, or a part of the gel film may adhere to the cover sheet above, so you can remove the cover sheet without damaging the gel film. Skill was required to remove it.

Also, when performing exposure operations in autoradiography after removing the cover sheet,
To prevent the photographic film from becoming wet, the gel must be dried or covered with a thin water-impermeable sheet, such as a plastic wrapping sheet, over the gel. This procedure of drying the gel has the advantage of increasing the sensitivity of the resulting autoradiography, but requires additional operational precautions due to the risk of damaging or contaminating the gel membrane during the procedure. It becomes necessary. On the other hand, with the method of covering with a thin sheet such as a plastic wrapping sheet, air bubbles may remain between the gel and the sheet.
The major drawback is that wrinkles often form on the sheet, which impairs the adhesion between the gel and the photographic film, resulting in blurred autoradiographs and reduced resolution.

The above-mentioned problems caused by removing the cover sheet during the exposure operation by autoradiography can be solved by the thickness described in JP-A-59-126237.
A solution has been attempted by using electrophoretic analysis materials with cover sheets that are 50 μm or less. In other words, by using the thin cover sheet mentioned above,
Exposure operations for autoradiography can be performed with the cover sheet still in place.

However, in consideration of the sensitivity and resolution of autoradiography, it is more desirable that the electrophoretic medium membrane used in the exposure operation in autoradiography be in a dry state during the operation.

The above problems are noticeable when using a gel membrane as a media membrane for electrophoresis, but membranes made of other materials, such as self-supporting porous membranes (e.g., cellulose acetate porous membrane, filter paper), etc. Similar problems arise when used as media membranes.

[Object of the Invention] An object of the present invention is to provide an electrophoretic medium material that is easy to handle during the operation of forming an electrophoretic medium membrane, during storage, during electrophoresis, and during the exposure operation in autoradiography. be.

Another object of the present invention is to provide an electrophoretic medium material that has excellent sensitivity and resolution in exposure operations using autoradiography.

[Summary of the Invention] The present invention provides an electrophoretic media material comprising an electrophoretic media membrane provided between a cover sheet and a support, wherein the cover sheet has an average pore diameter that crosses the plane of the sheet. The present invention relates to an electrophoresis medium material comprising a membrane filter having micropores (continuous micropores, ie, through holes) of 0.01 μm to 20 μm.

[Effects of the Invention] The electrophoresis medium material of the present invention has a cover sheet, which makes it easy to create an electrophoresis medium membrane, and the electrophoresis medium membrane is difficult to break during storage and electrophoresis. have Further, since the cover sheet of the electrophoretic medium material of the present invention is composed of an air-permeable microporous membrane filter having continuous micropores extending across the plane of the sheet, the electrophoretic medium membrane can be used for autoradiography. There is no need to remove the cover sheet during the drying operation. Therefore, the electrophoresis medium material of the present invention can be handled with the cover sheet provided at all times from the time of preparation to analysis operations.

In other words, the electrophoresis medium material of the present invention can be used even if foreign matter comes into contact with the surface of the electrophoresis medium membrane during preparation, storage, and electrophoresis, and it cannot be handled by holding parts of the electrophoresis medium membrane. There is also little risk of breaking the electrophoretic media membrane, and it is easier to handle than conventional electrophoretic media materials. Another advantage is that even if a sample is dropped somewhere other than the sample port during sample injection, it can be wiped off.

Further, in the electrophoresis medium material of the present invention, since the electrophoresis medium membrane is in a dry state during the exposure operation of autoradiography, the resulting autoradiograph has excellent sensitivity and resolution. The above effects of the present invention also mean that autoradiography failures can be further reduced. Normally, autoradiographic exposures take a long time, and if an exposure fails due to the problems mentioned above, the next exposure will take an even longer time than the previous one due to the decay of the radioisotope's radioactivity. Since it requires an exposure time of several hours, the time loss is very large.

Therefore, the simplification of autoradiography operations according to the present invention can greatly contribute to shortening experimental time. Furthermore, the electrophoresis medium material of the present invention preserves the electrophoresis membrane containing the sample in a dry state, so that the sample has excellent storage stability after electrophoresis. Especially when the sample is a protein, the electrophoresis medium material of the present invention can be stored for a long period of time. Furthermore, since the electrophoresis medium material of the present invention is in sheet form, it can be stored by stacking it,
It also has the advantage of not taking up much space.

[Detailed Description of the Invention] As an example of the structure of the electrophoresis medium material of the present invention, an electrophoresis medium membrane 2 as shown in FIG.
An example of this is a structure in which the cover sheet 1 and the support body 3 sandwich the cover sheet 1 and the support body 3.

The electrophoretic medium material of the present invention is characterized in that the cover sheet is made of a microporous sheet having continuous micropores extending across the plane of the sheet. In the present invention, "a microporous sheet having continuous micropores that cross the plane of the sheet" refers to fine (preferably average pore diameter of about 0.01 μm to about 20 μm, more preferably about 0.01 μm to about 10 μm) and continuous This means that a large number of hole-like portions consisting of voids are provided perpendicularly or substantially perpendicularly to the plane of the sheet, thereby forming a sheet-like structure with air permeability.

The continuous micropores provided in the cover sheet prevent the electrophoresis media membrane from being contaminated by microorganisms and particulates in the air during the preparation, storage, and analytical operations of the electrophoresis media material.
It is desirable that the pore diameter be sufficiently small.
In other words, the average pore diameter of the pores consisting of continuous voids is determined depending on the substance to be analyzed, storage conditions, analysis conditions, etc., but it is generally less than 20 μm, and is suitable for airborne bacteria and similar sizes. In order to completely prevent contamination from fine particles (i.e., to maintain sterility), the average pore diameter should be 1 μm or less, and if it is necessary to prevent the contamination of viruses or similar ultrafine particles, the average pore size should be 0.1 μm or less. preferable. Further, in order to completely prevent contamination by microorganisms and particulates, it is preferable that all continuous micropores have a substantially uniform pore diameter.

When the electrophoretic medium material of the present invention is used to dry an electrophoretic medium membrane with a cover sheet attached, the time required for the drying operation is most influenced by the porosity of the cover sheet. Therefore, in order to shorten the time required for the drying operation, it is necessary to use a cover sheet with a high porosity. In addition, porosity is determined by the number and average diameter of continuous micropores, so if it is necessary to reduce the average diameter of continuous micropores, the number of continuous micropores per unit area of the cover sheet should be reduced. There must be a sufficient number of holes. On the other hand, if the number of continuous micropores is too large (the porosity is too large), the cover sheet may lack strength and may be inconvenient or impossible to handle. Therefore, the porosity of the cover sheet is specifically about 25% to about 90%,
Preferably it is about 50% to about 85%.

The material of the cover sheet (membrane filter) is not particularly limited as long as it is possible to form the above-mentioned structure having continuous micropores during manufacturing.
If there is a risk that the cover sheet will undergo deformation such as distortion during the drying operation, it is preferable to use a hydrophobic material. Examples of hydrophobic materials include polyamides (nylons), vinyl chloride, fluorine-containing polymers (e.g., polytetrafluoroethylene, tetrafluoroethylene-hexafluoropropylene copolymers, polychlorotrifluoroethylene, etc.), polyethylene, and bisphenols. Examples include polycarbonate of A.

The structure of the cover sheet (membrane filter) having continuous micropores can be formed using various methods. For example, the above-mentioned cover sheet is manufactured by coagulating a colloidal solution, the sheet is immersed in a swelling solution to swell it to form continuous micropores, and the sheet is irradiated with neutron beams and then chemically etched. Examples include a method in which continuous fine pores are formed using a powder, and a method in which fine powder is formed into a sheet shape and heated and sintered.

For more information on membrane filters, see Robert E.
Synthetic Polymer Membrane (Synthetic Polymer Membrane)
Polymeric Membranes; 1971, McGraw-Hill Book Company
Company], US Pat. No. 1,421,341, and Japanese Patent Publication No. 53-21677, respectively. With reference to the membrane filter manufacturing methods described in these documents,
It is easy to manufacture the cover sheet of the present invention having characteristics such as average pore diameter, porosity, and material depending on storage conditions, analysis conditions, and the like. Moreover, various types of membrane filters are already commercially available from many manufacturers, and a membrane filter can be selected from among these commercially available products as necessary and used as the cover sheet of the present invention.

Specific examples of the above membrane filter include:
Examples include microporous polytetrafluoroethylene membranes and microporous polyethylene membranes.

The thickness of the cover sheet (D ¹ in Figure 1) is
If exposure for autoradiography is to be carried out through the cover sheet, it is preferred that it be as thin as possible. Specifically, a thickness of about 7 μm to about 200 μm is used, and the practically preferred thickness is about 10 μm.
to about 150 μm, and particularly preferred is a thickness in the range of about 20 μm to about 100 μm.

The support used in the electrophoresis medium material of the present invention is preferably a sheet-like material with good flatness, non-conductive and substantially water-impermeable.
Specific examples of the support include polyesters such as polyethylene terephthalate and bisphenol A polycarbonate, vinyl polymers such as polymethyl methacrylate, polyethylene, polystyrene, and polyvinyl chloride, and polyamides such as nylon. Examples include copolymers (eg, vinylidene chloride/vinyl chloride copolymer).

The thickness of the support (D ³ in Figure 1) is approximately
It is selected from the range of 5 μm to about 5 mm, preferably about 8 μm to about 3 mm. The thickness of the support may be the same as or different from that of the cover sheet, but it is preferred that the support is thicker and stiffer than the cover sheet.

As shown in FIG. 2, the cover sheet 1 and support body 3 are preferably sealed at both left and right ends with seals 6a and 6b. The width (L ³ ) for sealing is sufficient as long as the cover sheet 1 and support body 3 are fixed, and is preferably selected from a range of 2 mm to 20 mm. Alternatively, a cover sheet and a support may be adhered to a spacer, which will be described later, in place of a seal.
The other two sides may be sealed or left open.

There are no particular limitations on the electrophoretic medium membrane used in the electrophoretic medium material of the present invention. Typical materials for electrophoresis media membranes include:
Acrylamide gel, agarose gel, starch gel, agar gel, cellulose acetate porous membrane,
Examples include filter paper. Further, examples of the shape of the electrophoresis medium membrane include those shown in FIG. 1 (schematic cross-sectional view) and FIG. 2 (schematic plan view). The thickness (D ² ) of this electrophoresis medium membrane is selected depending on the purpose of separation, but is usually in the range of 50 μm to about 10 mm, preferably in the range of about 200 μm to about 5 mm in the case of a gel membrane. , and in the case of porous membranes and filter papers, it ranges from about 70 μm to about 1 mm. In addition, the size of the electrophoresis medium membrane (L ¹ + L ⁴ ) can be freely selected depending on the purpose, but especially in the case of gel membranes for DNA base sequencing, L ¹ is in the range of 20 cm to 40 cm, L ⁴ is 30cm
It is preferable that the range is from 60cm to 60cm.

As shown in FIGS. 2 to 5, spacers 5a and 5b are preferably provided on both outer sides of the electrophoresis medium membrane to maintain and protect the thickness of the electrophoresis medium membrane. It is desirable that the spacer is at least adhesively fixed to the support or integrated with the support. When the spacer and the support are integral, the spacer and the support can be manufactured integrally by a method such as molding. The thickness of the spacer is no greater than the thickness (D ² ) of the electrophoretic medium membrane 2. Further, the width L ² of the spacer is preferably selected within the range of 5 mm to 20 mm.

At one end of the electrophoresis medium membrane, there is a slot 4 (Figure 2) used as a sample injection port when performing vertical electrophoresis analysis, or a sample injection port when performing horizontal electrophoresis analysis. It is convenient to form the sample groove 7 (FIGS. 3 to 5) used as a sample in advance. Sample injection slot 4
The electrophoresis medium material provided with the slot may have a structure in which the entire slot is covered with a cover sheet, but the cover sheet covers the area where the sample can be injected and held in the part connected to the slot (the right side of slot 4 in Figure 2). It is also possible to have a structure in which the remaining part is exposed. Furthermore, in an electrophoresis medium material provided with a sample groove 7, the sample groove 7 may be covered with a cover sheet 1 (Fig. 3), or only that portion may be without a cover sheet ( Figures 4 and 5). However, a structure in which there is no cover sheet above the sample groove as shown in FIGS. 4 and 5 is preferable.
It is also possible to create a structure in which the cover sheet in the sample groove portion can be peeled off to open and close.

To create the electrophoretic medium material of the present invention,
For example, a method is used in which an electrophoretic medium film is formed on a horizontally placed support by a known method such as casting, coating, or adhesion, and then a cover sheet is pressed with a roller or spatula to laminate the film. be able to. The surfaces of the cover sheet and the support that come into contact with the electrophoresis medium membrane are
In order to improve the compatibility with the electrophoresis media membrane, known hydrophilic treatment methods (e.g., ultraviolet irradiation, glow discharge treatment, corona discharge treatment, electron beam irradiation, flame treatment, chemical etching, electrolytic etching, etc.) are used. It is preferable to heat it beforehand. In addition, when using a gel membrane as a medium membrane for electrophoresis, two sheets are pasted on a glass plate, etc., a mold is made using this, and the gel forming liquid is gelated in the mold. The electrophoretic medium material of the present invention can also be produced in this manner.

In addition, in order to prevent moisture evaporation in the electrophoresis medium membrane, drying such as sealing the appropriate sides around the electrophoresis medium membrane and the cover sheet (membrane filter) of the present invention with a heat seal or the like is performed. It is preferable to take preventive measures. As a measure to prevent the electrophoresis medium membrane from drying, there may be mentioned a method of storing the electrophoresis medium material in a bag made of moisture-impermeable paper or the like.

Next, examples of the present invention and comparative examples will be described.

[Example 1 and Comparative Example 1] Thickness with surface made hydrophilic by ultraviolet irradiation treatment
On a 175 μm colorless and transparent polyethylene terephthalate (PET) sheet (support), add 100 ml of electrophoretic medium film forming solution with the following composition, 1.3 ml of ammonium peroxodisulfate (10% by weight) as a polymerization initiator, and N,N. , N',N'-tetramethylethylenediamine (50μ) was molded to a thickness of 0.5 mm under nitrogen to obtain a polyacrylamide gel film.

Electrophoresis medium film forming solution : Acrylamide 9.5g N,N'-methylenebisacrylamide 0.5g Urea 42g Tris(hydroxymethyl)aminomethane in 100ml
1.08g Boric acid 0.55g EDTA・2Na salt 93mg Polytetrafluoroethylene (thickness 100 μm, average pore size
A 0.45 μm membrane filter) was placed thereon to prepare the electrophoresis medium material of the present invention.

For comparison, a polyacrylamide gel membrane was prepared using two glass cells and used as a comparative electrophoresis medium material. The following experiments were conducted using these two types of electrophoresis medium materials.

A sample of ³² P-labeled DNA subjected to Maxam-Gilbert digestion was subjected to electrophoretic analysis using the above two types of electrophoresis media materials, and the base sequence of the DNA was determined using autoradiography.

With the electrophoresis medium material of the present invention, the electrophoresis medium membrane could be dried under reduced pressure using a gel dryer without removing the cover sheet after electrophoresis.

On the other hand, drying the comparative electrophoresis medium material required removing the glass plate, and great care was required to perform this removal and drying operation.

After the above operations, autoradiography was performed on each electrophoresis medium material, and sharp separated images were obtained for all of them.

[Brief explanation of drawings]

FIG. 1 is a schematic cross-sectional view showing an example of the structure of the electrophoresis medium material of the present invention. FIG. 2 is a schematic plan view showing another example of the structure of the electrophoresis medium material of the present invention. Figures 3, 4, and 5 are schematic cross-sectional views (upper view) and schematic plan views (lower view) of various other configuration examples of the electrophoresis medium material of the present invention. be. 1...Cover sheet (membrane filter),
2... Electrophoresis medium membrane, 3... Support, 4... Sample injection slot, 5a, 5b... Spacer, 6a,
6b... Seal portion, 7... Sample groove, D ¹ ... Thickness of cover sheet (membrane filter), D ² ... Thickness of electrophoresis medium membrane, D ³ ... Thickness of support, L ¹
... Width of electrophoresis medium membrane, L ² ... Width of spacer,
L ³ ...Width of the seal part, L ⁴ ...Length of the electrophoresis medium membrane.

Claims (1)

[Scope of Claims] 1. An electrophoretic medium material in which an electrophoretic medium membrane is provided between a cover sheet and a support,
A medium material for electrophoresis, characterized in that the cover sheet is made of a membrane filter having micropores with an average pore diameter of 0.01 μm to 20 μm that extend across the plane of the sheet. 2. The electrophoresis medium material according to claim 1, wherein the membrane filter is a hydrophobic membrane filter. 3. The electrophoretic medium material according to claim 1, wherein the membrane filter is a membrane filter made of a fluorine-containing polymer.