JP5748121B2 - Separation and adsorption kit, separation and adsorption apparatus, and molecular analysis method - Google Patents

Description

  The present invention relates to a separation adsorption kit, a separation adsorption apparatus using the separation adsorption kit, and a molecular analysis method using the separation adsorption kit.

  As a technique for separating molecules to be analyzed by passing an electric current through a buffer solution through a separation medium, gel electrophoresis is used to differentiate molecules having a relatively large molecular weight, such as molecules in biological samples. It is widely used as a separation method based on this, and various methods and apparatuses have been developed so far for immobilizing and analyzing proteins or nucleic acids. For example, SDS-polyacrylamide gel electrophoresis using an anionic surfactant sodium dodecyl sulfate (SDS) is widely used for protein separation. Western blotting is well known as a method for analyzing and identifying separated proteins. Western blotting is a method for identifying proteins by antigen-antibody reaction by immobilizing proteins separated by electrophoresis by transferring them from a gel to a transfer film that is an adsorbing member. These separation and identification methods are extremely excellent in terms of specificity and detection sensitivity, and are widely used in the medical, pharmaceutical, biochemical, and food fields as highly reliable analytical techniques.

  On the other hand, various techniques for performing electrophoresis and transfer processes in a short time have been studied. For example, while performing electrophoresis, the transfer film is brought into contact with the gel exposed at the downstream end of the electrophoresis, and this contact state is maintained. A technique is disclosed in which molecules separated from a sample by electrophoresis are immediately and sequentially transferred to a transfer film by moving the transfer film (see Patent Document 1). This technique performs electrophoresis and transfer simultaneously, and is also suitable for automation.

In addition, techniques for improving the versatility of the above technique have been studied. For example, various electrophoresis kits for mounting on an electrophoresis apparatus for the purpose of simplifying operations have been disclosed. Yes.
As such an electrophoresis kit, after mounting on a fixture adjusted so that a rubber pad is arranged at the downstream end of the electrophoresis section, the gel is filled in the electrophoresis section, and then the fixation tool is removed, and then electrophoresis is performed. A device that is attached to an apparatus is disclosed (see Patent Document 2). This electrophoresis kit improves the moldability and storability of the gel from the time of gel filling to the time of mounting on the apparatus by using the fixture.
Further, a transfer film attached to a curved frame is disclosed (see Patent Document 3). By setting the transfer film to a state in which tension is applied by the frame, generation of wrinkles and deflection during transfer is suppressed, and transfer can be performed with high accuracy.

US Pat. No. 5,234,559 JP-T-9-501774 Japanese National Patent Publication No. 9-501239

  However, in the conventional electrophoresis kit, even after mounting on the electrophoresis apparatus, there is no material in which the exposed downstream end of the gel, which becomes a contact portion with the transfer film, is appropriately protected. . Since the contact part of the gel affects the transfer accuracy of the molecules separated from the sample onto the transfer film itself, it is important to prevent alteration and damage until just before use. In the conventional electrophoresis kit, From this point of view, none of the contact sites was appropriately protected.

  The present invention has been made in view of the above circumstances, a separation adsorption kit capable of protecting the contact portion of the separation medium with the adsorption member until the use immediately after being attached to the apparatus, and a separation adsorption apparatus using the separation adsorption kit It is another object of the present invention to provide a molecular analysis method using the separation and adsorption kit.

The present invention relates to a separation medium for separating molecules to be analyzed by passing an electric current through a buffer solution, a separation tank storing the separation medium, and a porous adsorption member for adsorbing the separated molecules from the separation medium. And the separation tank has first and second openings for flowing current, and the adsorption member protects the exposed surface of the separation medium. A separation adsorption kit is provided, wherein the adsorption member is arranged so that the protection portion is in close contact with the exposed surface of the separation medium in the second opening.
The present invention also provides a separation / adsorption kit characterized in that, in such a separation / adsorption kit, the protection part is connected to the adsorption member.
The present invention also provides a separation / adsorption kit characterized in that, in such a separation / adsorption kit, the protection part is attached to the adsorption member.
The present invention also provides a separation / adsorption kit characterized in that, in such a separation / adsorption kit, a part of the adsorption member is altered to form the protection part.
The present invention also provides a separation / adsorption kit, wherein the protection part is bonded to the second opening.
The present invention also provides a separation / adsorption kit, wherein a conductive medium that allows the molecules to pass through is provided between the second opening and the protection part. To do.
The present invention also provides a separation / adsorption device comprising such a separation / adsorption kit, further comprising a drive means for moving the adsorption member in the separation / adsorption kit. .
The present invention also provides a molecular analysis method using such a separation / adsorption kit, wherein the adsorption member is moved, the protection unit is moved from the second opening, and the separated molecules are separated from the separation medium. There is provided a method for analyzing molecules, comprising a step of adsorbing the adsorption member.

  According to the present invention, the separation adsorption kit that can protect the contact portion of the separation medium with the adsorption member immediately after use in the apparatus, the separation adsorption apparatus using the separation adsorption kit, and the separation adsorption kit are used. A molecular analysis method can be provided.

It is the schematic which shows the specific example of the kit for electrophoresis which concerns on one Embodiment of this invention, (a) is a perspective view, (b) is sectional drawing in the AA of the gel filling tank of (a), ( c) is an exploded view of the gel filling tank. FIG. 2 is a front view of a composite transfer film in the electrophoresis kit shown in FIG. 1. It is a schematic perspective view which illustrates the connection method with the drive means of the drive auxiliary member shown to FIG. It is a front view which shows the specific example of the comb used at the time of gel preparation. It is the schematic which shows the specific example of the kit for electrophoresis which concerns on other embodiment of this invention, (a) is a perspective view, (b) is an exploded view, (c) is the electrophoresis of the gel filling tank in (b). It is an expanded sectional view of a downstream end. It is an enlarged view in the area | region containing the 1st connection part and 2nd connection part of a composite transfer film. It is the schematic which shows the other specific example of the main body of a cathode buffer solution tank suitable for the kit for electrophoresis, (a) is a perspective view, (b) is sectional drawing in the BB line of (a). . It is a schematic sectional drawing for demonstrating the specific example of the analysis method of the molecule | numerator which concerns on one Embodiment of this invention.

<Separation adsorption kit>
The separation / adsorption kit according to the present invention adsorbs separated molecules from the separation medium, a separation medium that separates molecules to be analyzed by passing an electric current through a buffer, a separation tank that stores the separation medium, and the separation medium. A separation adsorbing kit comprising a porous adsorbing member, wherein the separation tank has first and second openings for passing an electric current, and the adsorbing member exposes the separation medium. A separation part according to the present invention, wherein a protection part for protecting the surface is provided, and the adsorption member is arranged so that the protection part comes into close contact with the exposed surface of the separation medium in the second opening. The adsorption kit is not particularly limited as long as it has the above-described configuration. As a preferred embodiment, an electrophoresis kit can be exemplified. Such an electrophoresis kit includes a gel filling tank and a composite transfer film. The gel filling tank is a part for separating the molecule to be analyzed from the sample by gel electrophoresis, the downstream end of the electrophoresis is opened, and the filled gel is exposed at the opening. The composite transfer film includes a transfer film and a protective sheet connected on the end side. The transfer film transfers the separated molecules from the gel, and the protective sheet adheres to the exposed surface of the gel at the opening and covers the gel for protection. Hereinafter, the electrophoresis kit will be described in detail with reference to the drawings.

  In the present embodiment, the molecule to be analyzed is usually a polymer compound, and is preferably a biological compound or a biologically relevant polymer compound, such as a protein, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid, Examples include sugars and complexes formed by combining or interacting two or more of these. It may be naturally derived, artificially synthesized, or a complex of these. In the case of natural origin, it may be derived from any of animals, plants and microorganisms.

FIG. 1 is a schematic diagram illustrating a specific example of an electrophoresis kit according to the present embodiment, in which (a) is a perspective view, (b) is a cross-sectional view taken along the line AA of the gel filling tank of (a), (C) is an exploded view of a gel filling tank.
The electrophoresis kit 1 shown here includes a gel filling tank 2 filled with a gel, and a composite transfer film 3 in which a protective sheet 32 is connected to a transfer film 31.

  The gel filling tank 2 is composed of an upper plate 21, a lower plate 22 and a spacer 23. More specifically, the two spacers 23 are disposed at the peripheral portions of the upper plate 21 and the lower plate 22 facing each other, with the upper surface thereof being in close contact with the upper plate 21 and the lower surface thereof being respectively in contact with the lower plate 22. A gel filling part 20 for filling the gel for electrophoresis is formed. The gel is filled (stored) in the gel filling unit 20, but is not shown in FIG. And the gel filling tank 2 has the 1st opening part 2a, the 2nd opening part 2b, and the 3rd opening part 2c. The first opening 2a is located on the upstream side of the migration, and the second opening 2b is located on the downstream side of the migration. In the present invention, “upstream migration” refers to upstream in the migration direction of the sample, and “downstream migration” refers to downstream in the migration direction of the sample. Accordingly, the downstream end of the migration is a site for transferring the molecules separated from the sample to the transfer film. Further, in FIG. 1 (a), the gel filling tank 2 and the protective sheet 32 are separately shown in order to make the configuration of the electrophoresis kit 1 easy to understand. However, as will be described later, these are the second openings. The part 2b is in close contact with the exposed surface of the gel.

The upper plate 21 is provided with a third opening 2c in the vicinity of the peripheral edge along the peripheral edge on the first opening 2a side. The 3rd opening part 2c corresponds to the injection port of the raw material for gel mentioned later. The third longitudinal direction of the length Y ₁₁ of the opening 2c is same as either to the long side direction of the length of the first opening 2a, or longer is preferred.
The size of the upper plate 21 may be appropriately adjusted according to the number and amount of samples to be analyzed. However, from the viewpoint of handleability, the length of the long side (the length in the direction orthogonal to the migration direction). ) Y ₁ is preferably 40 to 80 mm, the short side length (length in the migration direction) X ₁ is preferably 20 to 40 mm, and the thickness Z ₁ is 0.5 to 2 mm. Is preferred.

  The lower plate 22 is the same as the upper plate 21 except that the third opening 2c is not provided, and its shape (except that the third opening 2c is not provided) and size are large. The height is preferably the same as the upper plate 21 to be combined.

The spacer 23 has a plate shape or a rod shape, and may be adjusted in size so that the gel filling portion 20 has a desired width. For example, it from the viewpoint of handling property, that the length of the long sides (the length of the direction of electrophoresis) X ₂ is in the range similar to the X ₁ is preferably the same as X ₁ in combination upper plate 21 preferable. Further, the length of the short side (the length in the direction orthogonal to the migration direction) Y2 is preferably ₂ to 5 mm. Then, it is preferable that the thickness Z ₂ is the same range as with the Z _1.

The material of the upper plate 21, the lower plate 22 and the spacer 23 only needs to have insulating properties, such as glass such as quartz glass; ceramics; acrylic resin, polycarbonate, polystyrene, polyethylene terephthalate (PET), polyvinyl chloride, etc. A synthetic resin can be exemplified. Among these, glass is preferable because it has hydrophilicity and is excellent in flatness and heat dissipation.
And the upper board 21, the lower board 22, and the spacer 23 should just be produced by a well-known method according to the material.

  The gel filling tank 2 is preferably prepared by fixing the upper plate 21, the lower plate 22 and the spacer 23 to each other by a known method such as a method of using an adhesive or a method of engaging the gel filling bath.

  The gel filling tank 2 is merely illustrated as a preferable example in the electrophoresis kit 1, and the gel filling tank in the present embodiment is not limited thereto, and the molecule to be analyzed can be separated from the sample by gel electrophoresis. Any material can be used as long as it can be transferred.

  The composite transfer film 3 is obtained by connecting a transfer film 31 and a protective sheet 32 on each end side. In addition, FIG. 2 shows a front view of the composite transfer film 3 in the electrophoresis kit 1. Reference numeral 30 a indicates a connecting portion (hereinafter abbreviated as “first connecting portion”) between the transfer film 31 and the protective sheet 32. Furthermore, the end portion side of the protective sheet 32 opposite to the first connecting portion 30 a is connected to the end portion side of the drive assisting member 33. Reference numeral 30 b indicates a connecting portion (hereinafter abbreviated as “second connecting portion”) between the protective sheet 32 and the drive assisting member 33.

The transfer film 31 is for transferring and fixing molecules separated from the sample by electrophoresis from the gel.
The protective sheet 32 is in close contact with the exposed surface at the downstream end of electrophoresis until the start of electrophoresis, thereby protecting the gel.
The driving assisting member 33 connects driving means for moving the transfer film 31 and the protective sheet 32 in the connecting direction in the electrophoretic migration.
In the composite transfer film 3, the connecting direction (longitudinal direction of the composite transfer film 3) of the transfer film 31, the protective sheet 32, and the drive assisting member 33 matches the migration direction of the sample (the arrow direction in FIGS. 1 and 2). And moved in this direction during electrophoresis.

  The material of the transfer film 31 is not particularly limited as long as it is a porous material capable of adsorbing molecules, and may be a known material. For example, when the molecule to be analyzed is a nucleic acid such as DNA or RNA, or a biomolecule such as protein, a preferable material is a synthetic resin such as polyvinylidene fluoride (PVDF) or nitrocellulose. For example, as a commercial product of a PVDF transfer membrane, Immobilon PVDF transfer membrane (manufactured by Millipore) can be exemplified.

The size of the transfer film 31 may be set according to the size of the gel filling tank 2 to be combined. For example, (in the direction of length perpendicular to the moving direction of the composite transfer layer 3) Y ₃ short side length of is preferably at least equivalent for the same length of the gel-filled portion 20, from the viewpoint of handling property, it is preferably the same range as the Y _1. (Moving length in the direction of the composite transfer layer 3) X ₃ long side length of, depending on the moving speed at the time of electrophoresis of the transfer film 31 is preferably 80 to 120 mm. The thickness of the transfer film 31 is preferably 0.05 to 2 mm.

  The material and thickness of the protective sheet 32 are preferably selected as appropriate according to the expected gel storage conditions until electrophoresis is performed. For example, a method of selecting so as to have one or more properties of gas barrier properties, liquid barrier properties, light barrier properties, and heat barrier properties.

For example, in order to suppress drying in a gel during storage, the protective sheet 32 may be liquid-impermeable.
Further, in the gel during storage, the protective sheet 32 has a thermal conductivity lower than the thermal conductivity of water in order to suppress deterioration due to a change in environmental temperature (temperature outside the electrophoresis kit 1). More specifically, the thermal conductivity is preferably less than 0.6 W / (m · K).
Moreover, in order to suppress deterioration due to light in the gel during storage, the protective sheet 32 preferably has a light transmittance of 40% or less, more preferably 20% or less, and less than 10%. It is particularly preferred. And since ultraviolet light is easier to denature the gel than visible light, it is preferable that the transmittance | permeability of ultraviolet light is the said range especially. As such a protective sheet 32 having a low light transmittance, a colored sheet is preferable, and a colored opaque sheet is more preferable.

  Examples of the material for realizing liquid impermeability, thermal conductivity, or light transmittance as described above include polyethylenes such as low density polyethylene (LDPE), medium density polyethylene, and high density polyethylene (HDPE); polypropylene ( Polypropylene (PS); Polyesters such as polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene naphthalate (PEN), polybutylene naphthalate (PBN); polyvinyl chloride (PVC); polyvinylidene chloride (PVDC); acrylic resin; metallocene polyethylene ethylene / vinyl acetate copolymer; unstretched K (PVDC) coat resin; K coat polyester stretched nylon; K coat nylon; Vinyl alcohol); cellophane; polyacrylonitrile (PAN); polyurethane; natural rubber; acrylic rubber (ACM, ANM), nitrile rubber (NBR), isoprene rubber (IR), chloroprene rubber (CR), ethylene propylene rubber (EPM, EPDM) ), Styrene-butadiene rubber (SBR), butadiene rubber (BR), epichlorohydrin rubber (CO, ECO), silicone rubber, fluorine rubber (FKM), synthetic rubber such as butyl rubber; polytetrafluoroethylene (PTFE), poly Chlorotrifluoroethylene (PCTFE), polyvinylidene fluoride (PVDF), polyvinyl fluoride (PVF), perfluoroalkoxy fluororesin (tetrafluoroethylene perfluoroalkyl vinyl ether copolymer, PFA), tetrafluoroethylene -Hexafluoropropylene copolymer (FEP), ethylene-tetrafluoroethylene copolymer (ETFE), ethylene-chlorotrifluoroethylene copolymer (ECTFE), tetrafluoroethylene-perfluorodioxole copolymer A fluororesin such as (TFE / PDO) can be exemplified.

  The protective sheet 32 may be either a single layer or a plurality of layers. In the case of a plurality of layers, the materials of all layers may be the same, some may be different, or all may be different. And when at least one part is different, the combination of the material of these multiple layers can be set arbitrarily. For example, a plurality of materials having different properties such as a material having low liquid permeability, a material having low thermal conductivity, and a material having low light transmittance may be combined.

  The thickness of the protective sheet 32 may be appropriately adjusted according to the material and purpose, but is usually preferably 0.5 to 10 mm, and more preferably 1 to 7 mm. By setting it to the lower limit value or more, a higher effect can be obtained in which the gel is protected from external force and the deformation and breakage of the gel are suppressed. Moreover, handleability improves more by setting it as below an upper limit. When the protective sheet 32 has a plurality of layers, the thickness of the protective sheet 32 refers to the total thickness of these multiple layers.

The protective sheet 32 is preferably sized to cover the entire exposed surface of the gel. For example, (in the direction of length perpendicular to the moving direction of the composite transfer layer 3) Y ₄ length of the long side is preferably the same range as the Y _3, linked Y of the transfer film 31 ₃ may be the same. (Moving length in the direction of the composite transfer layer 3) _{X 4} length of the short side is preferably 10 to 50 mm. By setting the lower limit value or more, the exposed surface of the gel can be easily and reliably covered. Moreover, handleability improves more by setting it as below an upper limit.

  The connection method of the transfer film 31 and the protective sheet 32 may be appropriately selected according to these materials. As a preferable coupling method, the transfer film 31 and the end side of the protective sheet 32 are overlapped with each other, and a welding method in which the overlapped surfaces are heat-welded with each other, or a pasting in which the overlapped surfaces are bonded with an adhesive or a double-sided adhesive tape. Examples of the method include joining by a method such as a method and using the overlapping portion as the first connecting portion 30a. In the above means, commercially available products can be used for the thermal welding machine, the adhesive, and the double-sided pressure-sensitive adhesive tape, and the first connecting portion 30a can be formed according to a known method.

  Here, an example is shown in which the entire overlapping portion of the transfer film 31 and the protective sheet 32 is joined to form the first connecting portion 30a, but even if only a part of the overlapping portion is joined. Good. However, it is preferable that the entire region is joined from the viewpoint that the strength of the first connecting portion is improved and the transfer surface of the transfer film 31 can be widened.

(Moving length in the direction of the composite transfer layer _{3) X 31} connecting the width of the first connecting portion 30a is preferably 3 to 8 mm. By setting it as more than a lower limit, the transfer film 31 and the protection sheet 32 can be connected more stably, and handling property improves more by setting it as an upper limit or less. And it is preferable that the 1st connection part 30a is formed over the whole region of the direction orthogonal to the moving direction of the composite transfer film 3 of the transfer film 31 and / or the protection sheet 32. FIG.

  In addition, the thickness of the first connecting portion 30a (the thickness of the overlapping portion) is preferably 10 mm or less, and more preferably 5 mm or less. By setting it as such a range, the 1st connection part 30a of the state closely_contact | adhered to the said exposed surface of a gel can be moved more easily at the time of the movement of the composite transfer film 3. FIG. The lower limit of the thickness of the 1st connection part 30a is not specifically limited, What is necessary is just to set so that the 1st connection part 30a may have moderate intensity | strength.

The drive assisting member 33 has a belt-like shape, and one connection hole 33a serving as a connection portion with the drive means for moving the composite transfer film 3 is provided on each end of the longitudinal direction.
And the drive auxiliary member 33 can be connected by using the drive means 4 provided with the connection part 42, for example, as shown in FIG. Here, as the driving means 4, only the action portion 41 that is connected to the drive assisting member 33 and applies the driving force is mainly illustrated, but the action portion 41 is in the vertical direction (the moving direction indicated by the arrow in the drawing). The configuration is not particularly limited as long as it is movable.

  The drive unit 4 includes an action part 41 and connection members 42 and 42. The action part 41 is a rod-shaped member having a square cross section, and two concave parts 41a and 41a are provided on both ends in the longitudinal direction. Each of the connecting members 42 and 42 is a small square piece, and convex portions 42a and 42a having a square cross section extend from the center thereof. Then, the drive assisting member 33 is connected to the drive means 4 by fitting the protrusions 42 a, 42 a through the connection holes 33 a, 33 a of the drive assisting member 33 and the recesses 41 a, 41 a of the action portion 41. In addition, the cross-sectional shape of the action part 41 and the connection member 42 is not limited to what is shown here, It can set arbitrarily.

  The drive assisting member 33 moves in the same direction as the action portion 41 moves upward in the direction of the arrow in FIG. Thus, it is preferable that the drive assisting member 33 has a plurality of connection portions with the drive unit 4 in a direction (longitudinal direction) perpendicular to the moving direction of the composite transfer film 3. By doing so, the composite transfer film 3 can be moved with higher accuracy.

  The material of the drive assisting member 33 is not particularly limited as long as it has strength that allows the transfer film 31 and the protective sheet 32 to be moved together and has insulating properties. For example, if it is resin, it can select from what was mentioned as a material of the protection sheet 32 suitably.

The size of the drive assisting member 33 is not particularly limited as long as the composite transfer film 3 can be moved stably. Here exemplifies those strip (elongated sheet) as the auxiliary drive member 33, for example, the length of the long side (the direction perpendicular to the length of the moving direction of the composite transfer layer 3) Y ₅ is the Y ₄ is equivalent to the (moving length in the direction of the composite transfer layer 3) X ₅ length of the short sides may be equal to the X _4. Moreover, since the drive assisting member 33 does not come into contact with the gel, it is preferable that the thickness of the drive assisting member 33 is equal to or greater than the thickness of the protective sheet 32 from the viewpoint of providing sufficient strength. .

The connection method of the protection sheet 32 and the drive assisting member 33 may be appropriately selected according to these materials, and may be the same as the connection method of the transfer film 31 and the protection sheet 32, for example. The connection width (composite transfer direction of movement of the length of the membrane 3) X ₃₂ of the second coupling portion 30b may be similar to connecting a width of the first connecting portion 30a. The second connecting portion 30b is preferably formed over the entire region of the protective sheet 32 and / or the drive assisting member 33 in the direction orthogonal to the moving direction of the composite transfer film 3. By doing so, the composite transfer film 3 can be moved with higher accuracy.

  Here, an example is shown in which the entire overlapping region of the protective sheet 32 and the drive assisting member 33 is joined to form the second connecting portion 30b, but only a part of the overlapping region is joined. Also good. However, it is preferable that the entire region is joined from the viewpoint that the strength of the second connecting portion is improved and the protective surface of the protective sheet 32 can be widened.

  The drive assisting member 33 is not limited to the one shown here, and a part of the configuration may be changed as appropriate without departing from the effect of the present invention. For example, the drive assisting member 33 may have a clip-like structure, and the end portion side of the protective sheet 32 may be sandwiched and connected.

  Here, as the composite transfer film 3, the transfer film 31, the protective sheet 32, and the drive auxiliary member 33 are illustrated as being connected in this order in the moving direction (migration direction) of the composite transfer film 3. Can be omitted. In this case, for example, the connection hole corresponding to the connection hole 33a in the region including the end portion side of the protective sheet 32 opposite to the first connection portion 30a, for example, the portion corresponding to the second connection portion 30b. May be provided directly and connected to the driving means 4 here. Thus, although the drive assisting member 33 is not an essential configuration, the drive assisting member 33 is used in that the damage to the protective sheet 32 can be reliably suppressed and the composite transfer film 3 can be moved more easily. It is preferable to do.

  What is necessary is just to select suitably the gel with which the gel filling tank 2 was filled according to the kind of molecule | numerator of analysis object. Preferred examples of the gel include polyacrylamide gel and agarose gel. The gel may be used in combination of two or more materials, and the combination in that case can be arbitrarily selected. For example, in the case of a polyacrylamide gel, the concentration may be changed to have a two-layer structure of a separation part and a concentration part.

  In addition to the gel filling tank 2 and the composite transfer film 3 filled with the gel, the electrophoresis kit 1 may include other members, instruments, chemicals, or the like used during electrophoresis or before electrophoresis.

  For example, the electrophoresis kit 1 is preferably packaged with various packaging materials and stored in a sealed state. As the packaging material, those having one or more properties of gas barrier properties, liquid barrier properties, light barrier properties and heat barrier properties are preferable, and preferable specific materials include those similar to the protective sheet 32.

The electrophoresis kit 1 can be prepared by arranging the composite transfer film 3 so that the protective sheet 32 is in close contact with the exposed surface of the gel in the second opening 2b and filling the gel filling tank 2 with the gel. .
At the time of gel filling, for example, the gel raw material is injected from the third opening 2c of the gel filling tank 2, the comb is inserted into the third opening 2c, the gel raw material is polymerized as it is, and finally the comb Just pull through.

  FIG. 4 is a front view showing a specific example of the comb, and the comb 91 shown here has a comb shape having six convex portions 91a,... On one end face. In the electrophoresis kit 1, after the gel raw material is injected into the gel filling part 20 of the gel filling tank 2, the comb 91 is gelled from the comb teeth (convex parts 91 a,. By inserting into the third opening 2c of the filling tank 2 and polymerizing and gelling the gel raw material, the six convex portions 91a,. A comb-shaped shape in which concave portions (not shown) are continuously provided can be formed in the gel. In addition, the number of the convex parts of the comb for forming the sample introduction part can be arbitrarily set according to the purpose.

  Here, the composite transfer film 3 is shown in which a protective sheet 32 is connected to the transfer film 31. However, depending on the material of the transfer film 31, a part of the transfer film 31 may be altered to form a protective sheet. The composite transfer film may be used. Examples of the method for modifying the transfer film 31 include a method for changing the properties of the transfer film 31 by heating or organic solvent treatment. The method of altering the transfer film 31 is suitable, for example, for crushing the voids of the transfer film 31 and eliminating the porous property to form a liquid-impermeable protective sheet.

FIG. 5 is a schematic view showing a specific example of an electrophoresis kit according to another embodiment, (a) is a perspective view, (b) is an exploded view, and (c) is a gel filling tank 2 in (b). It is an expanded sectional view of the electrophoresis downstream end.
The electrophoresis kit 10 </ b> A shown here uses the electrophoresis kit 1 shown in FIG. 1, and is configured in combination with the cathode buffer solution tank 6 </ b> A.
The gel filling tank 2 of the electrophoresis kit 1 is filled with an electrophoresis gel 200. The composite transfer film 3 is arranged such that the connecting direction of the transfer film 31, the protective sheet 32, and the drive assisting member 33 (longitudinal direction of the composite transfer film 3) is along the sample migration direction indicated by the arrows in the figure. ing. Here, the protective sheet 32 is brought into close contact with the exposed surface of the filled gel 200 through the conductive liquid permeable sheet 5 in the second opening 2b located on the downstream side of the gel filling tank 2. Yes.

  For example, the protective sheet 32 is made of an adhesive in the portion of the second opening 2b where the gel 200 is not exposed (any one of the upper plate 21, the lower plate 22, and the spacer 23 of the gel filling tank 2). It is preferable that it is fixed to the gel filling tank 2 so as to be easily peelable. Here, “easily peelable” means that the protective sheet 32 is fixed with a weak adhesive force to such an extent that the protective sheet 32 is easily peeled off from the gel filling tank 2 as the composite transfer film 3 starts to move. The fixing portion of the protective sheet 32 in the second opening 2b is preferably a portion where the molecule of the transfer film 31 is transferred or the region to be transferred does not pass when the composite transfer film 3 moves. More specifically, the region of the end portion in the longitudinal direction of the second opening 2b, which is indicated by reference numeral 20b in FIG. 5B, can be exemplified. Here, the protective sheet 32 is fixed to the gel filling tank 2 via the liquid permeable sheet 5, but when the liquid permeable sheet 5 is not used, it is directly fixed to the gel filling tank 2. It may be.

  The adhesive for fixing the protective sheet 32 is preferably a water-resistant silicone adhesive or a resin adhesive. And in order to adjust adhesive force weakly, it is preferable to apply | coat the said adhesive agent in a spot shape in the said fixed part.

  The liquid permeable sheet 5 covers the second opening 2b of the gel filling tank 2 so as to cover the entire second opening 2b. And it is preferable that it fixes to the gel filling tank 2 with an adhesive or an adhesive etc. in parts other than the opening surface of the 2nd opening part 2b. By disposing the liquid-permeable sheet 5 in this way, the gel material can be polymerized more uniformly when the injected gel raw material is polymerized in the gel filling tank 2 and further separated. When the transferred molecules are transferred and adsorbed onto the transfer film 31, a high effect of suppressing damage due to friction between the gel 200 exposed at the second opening 2b and the transfer film 31 can be obtained. The liquid permeable sheet 5 is not an essential component, and the transfer film 31 may be directly adhered to the gel 200. However, by using the liquid permeable sheet 5, the above-described effects can be obtained, and molecular transfer can be performed. Can be performed with higher accuracy.

The liquid permeable sheet 5 may be any material that has conductivity and liquid permeability and allows the molecules to be analyzed to permeate. What consists of a material with low property is preferable. As a material having such properties, for example, when protein is analyzed, polyvinylidene fluoride (PVDF) can be exemplified, and a hydrophilic PVDF membrane (manufactured by Millipore) is commercially available.
The thickness of the liquid permeable sheet 5 is preferably 0.05 to 2 mm.

The cathode buffer tank 6 </ b> A is combined with the gel filling tank 2 and serves as a reservoir for the cathode buffer during electrophoresis, and includes a main body 61 and a sealing member 62.
The main body 61 has a shape in which a part of the side wall portion is removed in a rectangular container and is opened, and a stage 61b for installing the gel filling tank 2 is provided on the bottom portion 61a. And the side surface of the stage 61b protrudes from the main body 61 in the said open part.
The sealing member 62 has an L-shaped cross-section in a direction perpendicular to the longitudinal direction, and includes a side wall portion 62a and a bottom portion 62b perpendicular to the side wall portion 62a.

  The gel filling tank 2 integrated with the composite transfer film 3 is combined with the main body 61 of the cathode buffer tank 6A by placing the lower plate 22 on the stage 61b. Further, the sealing member 62 is provided with the outer surface of the bottom 62b on the upper plate 21 of the gel filling tank 2, so that the side wall 62a is connected to the side wall 61c of the main body 61 without a gap, A reservoir for the cathode buffer is formed. In addition, it is preferable that the main body 61 and the sealing member 62 of the cathode buffer tank 6A and the gel filling tank 2 are fixed to each other by a known method such as a method of engagement or a method of using an adhesive. The adhesive preferably has water resistance, more preferably a water resistant silicone adhesive or a resin adhesive, and a silicone-modified polymer adhesive Super X (produced by Cemedine) is an example of a commercially available product. it can.

  The composite transfer film 3 is disposed so that the transfer surface of the transfer film 31 is along the outer surface of the bottom 61a of the cathode buffer solution tank 6A. Thereby, during electrophoresis, the molecules separated from the sample can be continuously transferred from the gel 200 to the transfer film 31 by moving the composite transfer film 3. In the electrophoresis kit 10A, the gel filling tank 2 protrudes from the main body 61 together with the stage 61b, and the composite transfer film 3 can be easily moved when transferring molecules.

  FIG. 6 is an enlarged view of a region including the first connecting portion 30a and the second connecting portion 30b of the composite transfer film 3. Here, an example is shown in which the composite transfer film 3 is arranged so that the protective sheet 32 is located on the contact portion (the exposed surface of the gel 200) side with the gel 200 in the first connecting portion 30a. In the first connecting portion 30a, conversely, the transfer film 31 may be positioned on the contact portion side with the gel 200, but the arrangement form shown here is preferable. In the first connecting portion 30a, as shown here, a step due to the overlap of the transfer film 31 and the protective sheet 32 may occur. When the composite transfer film 3 is arranged as shown in the drawing, when the composite transfer film 3 moves in the analysis method described later, the exposed surface of the gel 200 is caused by the step in the moving direction (arrow direction in the figure). The first connecting portion 30a passes without being caught by the protruding portion. That is, the first connecting portion 30a passes more smoothly than when the composite transfer film 3 is arranged so that the transfer film 31 is positioned on the contact portion side with the gel 200.

  Here, in the second connecting portion 30b, an example is shown in which the driving assisting member 33 is located on the contact portion with the gel 200 (the exposed surface of the gel 200). The protective sheet 32 may be located on the contact portion side with the gel 200. Since the 2nd connection part 30b does not contact the gel 200 normally, the said exposed surface of the gel 200 is not influenced like the case of the 1st connection part 30a.

FIG. 7 is a schematic diagram illustrating another specific example of the main body of the cathode buffer solution tank suitable for the electrophoresis kit according to the present embodiment, in which (a) is a perspective view and (b) is a perspective view of (a). It is sectional drawing in the BB line.
The main body 63 of the cathode buffer tank 6B shown here has a convex curved surface in which the outer surface of the bottom 63a bulges outward. Further, the side surface of the stage 63b for installing the gel filling tank 2 is not projected from the main body 63, unlike the cathode buffer tank 6A. In the cathode buffer tank 6B, the other parts including the sealing member are the same as those of the cathode buffer tank 6A. Since the outer surface of the bottom 63a is a curved surface, the cathode buffer tank 6B facilitates the movement of the composite transfer film 3 during the transfer of molecules even when the side surface of the stage 63b is not projected as described above. It can be moved smoothly and safely. In addition, the side surface of the stage 63b may protrude as in the case of the cathode buffer solution tank 6A.

  The electrophoresis kit 10 </ b> A is not limited to the one shown here, and a part of the configuration may be appropriately changed within a range that does not hinder the effects of the present invention. For example, except for the electrophoresis kit 1, it can be configured in the same manner as a conventional electrophoresis kit. Further, the shape of the sealing member 62 is not particularly limited as long as the sealing member 62 has an installation site on the stage 61b and a side wall portion that can form a cathode buffer solution storage portion.

  As with the electrophoresis kit 1, the electrophoresis kit 10A is preferably packaged with various packaging materials and stored in a sealed state.

In the electrophoresis kit 10A, the composite transfer film 3 is disposed on the exposed surface of the gel 200 in the second opening 2b so that the protective sheet 32 is in close contact with the liquid permeable sheet 5 or directly. It can be produced by filling the tank 2 with the gel 200.
The filling method of the gel 200 is the same as that of the electrophoresis kit 1.

  Up to this point, the electrophoresis kits 1 and 10A have been described as the separation and adsorption kit according to the present invention. For example, depending on the molecule to be analyzed, a gel, a gel filling tank, a composite transfer membrane, and a liquid permeable sheet Instead, a combination of various separation media, separation tanks, adsorption members provided with protection units, and conductive media can be applied. That is, the separation medium and the adsorbing member may be appropriately selected according to the molecule to be analyzed, the protection unit may be appropriately selected according to the separation medium, and the conductive medium may be the molecule to be analyzed and the separation medium. What is necessary is just to select suitably according to. And the separation tank should just be the structure suitable for flowing an electric current through a buffer solution and isolate | separating this molecule | numerator.

<Separation adsorption apparatus and molecular analysis method>
The separation adsorption apparatus according to the present invention includes the separation adsorption kit, and further includes drive means for moving the adsorption member in the separation adsorption kit.
Further, the molecular analysis method according to the present invention is a molecular analysis method using the separation adsorption kit, wherein the adsorption member is moved, and the protection unit is moved and separated from the second opening. A step of adsorbing molecules from the separation medium to the adsorption member.
As a preferable embodiment of the separation and adsorption apparatus and analysis method, those using the electrophoresis kit can be exemplified.

  In the molecular analysis method according to the present embodiment, the transfer film is brought into close contact with the gel exposed surface from the state where the composite transfer film of the electrophoresis kit is moved and the protective sheet is in close contact with the gel exposed surface. Thus, the method includes the step of transferring the molecule to be analyzed from the gel to the transfer film. Hereinafter, a method for analyzing such molecules will be described in detail with reference to the drawings.

FIG. 8 is a schematic cross-sectional view for explaining a specific example of the molecular analysis method according to the present embodiment. Here, an analysis method using an electrophoresis apparatus 100A provided with an electrophoresis kit 10A as a separation and adsorption apparatus will be described.
FIG. 8A is a schematic cross-sectional view illustrating an electrophoresis device before starting electrophoresis. As shown in FIG. 8A, the electrophoresis apparatus 100A includes an electrophoresis kit 10A, a container 92, a DC power supply 93, electrodes 94 (cathode 94a and anode 94b), electric wires 95, and driving means 4. Has been.

  The accommodating portion 92 corresponds to a main body that performs electrophoresis, and a partition 920 includes a cathode-side accommodating portion 921 that includes a reservoir for the cathode buffer 99a, and an anode-side accommodating portion 922 that constitutes a reservoir for the anode buffer 99b. It is divided into.

In the cathode side housing portion 921, an electrophoresis kit 10A is disposed. Then, while maintaining the state in which the protective sheet 32 is in close contact with the exposed surface of the gel 200, the transfer surface of the transfer film 31 is along the outer surface of the side wall portion 61c and the bottom portion 61a of the cathode buffer solution tank 6A. A composite transfer film 3 is disposed. The composite transfer film 3 is further connected to the drive means 4 via a drive assisting member 33. A cathode buffer 99a is stored in the cathode buffer tank 6A.
On the other hand, the anode buffer 99b is stored in the anode housing portion 922.
In FIG. 8, for the sake of convenience, the first connecting portion 30a and the second connecting portion 30b are each indicated by a black dot “•”. Further, the illustration of the liquid permeable sheet is omitted.

  The partition wall 920 not only partitions the housing portion 92 but also has a function of bringing the protective sheet 32 and the transfer film 31 of the composite transfer film 3 into close contact with the exposed surface of the gel 200 in the second opening 2b. In addition, the partition wall 920 has an opening 920a, and thus the electrophoresis proceeds smoothly.

  The material of the partition 920 should just be what does not change with various buffer solutions. In order to improve the adhesion of the protective sheet 32 and the transfer film 31 to the gel 200, the partition wall 920 is preferably made of an elastic material. Examples of such a material include natural rubber and synthetic rubber. Specifically, what was illustrated as a material of the protection sheet 32 is mentioned.

The electrode 94 includes a cathode 94 a and an anode 94 b, and is electrically connected to a DC power source 93 by an electric wire 95.
The cathode 94a and the anode 94b can be made of a known material. Specifically, various metals, alloys and carbons can be exemplified, but platinum is preferable from the viewpoint of stability.

  As the cathode buffer 99a and the anode buffer 99b, those having a known composition can be used, and may be appropriately selected according to the type of molecule to be analyzed. Preferred are Tris / glycine buffer, Tris / glycine / SDS buffer, acetate buffer, sodium carbonate buffer, N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer, Tris / boron. Examples include acid / ethylenediaminetetraacetic acid (EDTA) buffer, Tris / acetic acid / EDTA buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, phosphate buffer, Tris / tricine buffer, Tris / methanol buffer, etc. it can.

  In the electrophoresis kit 10A, the exposed surface of the gel 200 (the contact portion of the gel 200 with the transfer film 31) is protected from the time of its preparation until immediately before the start of electrophoresis as shown in FIG. Since the sheet 32 is in close contact, the exposed surface is protected, and the gel 200 is prevented from being altered or damaged until just before use. Then, by appropriately selecting the material and thickness of the protective sheet 32, for example, prevention of drying, prevention of alteration due to changes in environmental temperature, prevention of alteration due to light, and prevention of deformation or damage due to external force. The target protective action such as one or more can be strengthened.

After setting the electrophoresis apparatus 100A, a sample is introduced into the gel 200 in the gel filling tank 2.
Next, the sample is electrophoresed and the composite transfer film 3 is moved so that the transfer film 31 is in close contact with the exposed surface of the gel 200 from the state in which the protective sheet 32 is in close contact with the exposed surface of the gel 200. . Due to the movement of the composite transfer film 3, the transfer film 31 and the protective sheet 32 move integrally in the connecting direction of these (composite transfer film 3), and the protective sheet 32 and the transfer film 31 sequentially or directly on the exposed surface of the gel 200. It adheres via the liquid-permeable sheet 5. Here, “the direction in which the transfer film 31 and the protective sheet 32 are connected” refers to the anode-side container 921 to the anode along the outer surface of the bottom 61a of the cathode buffer tank 6A immediately below the cathode buffer tank 6A. It is a direction toward the side housing portion 922, and is an upper side (here, the upper side in the vertical direction) along the opening surface of the second opening 2 b of the gel filling tank 2 on the exposed surface of the gel 200.

  The composite transfer film 3 moves by actuating the driving unit 4 and moving the action part 41. Then, by moving the composite transfer film 3, the transfer film 31 moves along the outer surfaces of the side wall portion 61c and the bottom portion 61a of the cathode buffer solution tank 6A. Furthermore, instead of the protective sheet 32, the transfer and adsorption of molecules can be performed from the stage when the transfer film 31 comes into contact with the exposed surface of the gel 200. FIG. 8B shows a state in which the transfer film 31 is in close contact with the exposed surface of the gel 200.

  Here, although the example which moves the action part 41 of the drive means 4 to the perpendicular direction upper side is shown, the movement direction of the action part 41 is a predetermined angle (it is not 0 degree with respect to the vertical direction in FIG. 8, for example) It may be a diagonally upward direction that is shifted by (angle), that is, a diagonally upward direction that is inclined in either direction of the cathode side accommodating portion 921 or the anode side accommodating portion 922.

  The movement start time of the composite transfer film 3 and the start time of electrophoresis can be arbitrarily set as long as the transfer of molecules to the transfer film 31 is not inhibited, either of which may be earlier or simultaneously.

  The composite transfer film 3 is preferably moved by automatically operating the driving means 4 according to a set program.

  The moving speed of the composite transfer film 3 is not particularly limited, and may be set in consideration of electrophoresis conditions. However, at the stage where the exposed surface of the gel 200 is in contact with the protective sheet 32, it is preferably 10 to 1000 μm / second. By doing in this way, the higher effect which protects the said exposed surface of the gel 200 is acquired.

  Electrophoresis may be performed under normal conditions in consideration of the type of molecule. For example, in order to suppress heat generation, the gel may be cooled with a Peltier element.

In the introduced sample, molecules contained by electrophoresis are gradually separated, and the separated molecules are discharged from the migration end (exposed surface) of the gel 200 located in the second opening 2b. Then, it is transferred to the transfer film 31 and fixed (adsorbed) for each molecule. At this time, since the gel 200 is prevented from being altered or damaged, the transfer accuracy of molecules to the transfer film 31 is high. When the liquid permeable sheet 5 is used, the protection effect of the gel 200 during the movement of the transfer film 31 is improved, so that the transfer accuracy is further increased. Here, “high transfer accuracy” means that, for example, a molecule can be transferred with high resolution and good reproducibility.
Further, by connecting the protective sheet 32 and the transfer film 31 to the driving means 4 via the driving auxiliary member 33, damage to the protective sheet 32 can be reliably suppressed and the composite transfer film 3 can be moved more easily. Can do.

  For example, after the movement of the composite transfer film 3 is stopped in the state shown in FIG. 8C after the electrophoresis and the adsorption of the molecules are completed, the composite transfer film 3 is removed from the electrophoresis apparatus 100A, and the transfer film 31 is removed. The target molecule can be analyzed by washing with pure water or the like as necessary and then subjecting it to various analyses. Here, as an analysis method, a known method such as western blotting, southwestern blotting, far western blotting, mass spectrometry, fluorescent staining, silver staining or the like may be arbitrarily applied depending on the type and purpose of the molecule. At this time, since the transfer is performed with high accuracy, information on molecules can be obtained with high accuracy.

  The analysis method according to the present embodiment can be performed by automatically automating the separation and adsorption of molecules by using the electrophoresis kit 10A. Further, by consistently automating from installation of the electrophoresis kit 10A to analysis, a higher effect of protecting the exposed surface of the gel 200 can be obtained.

  The analysis method according to this embodiment can also be applied to the transfer and adsorption of molecules in two-dimensional electrophoresis. For example, after separating proteins by isoelectric focusing, SDS-polyacrylamide gel electrophoresis is performed, and SDS-poly after isoelectric focusing in two-dimensional electrophoresis in which molecules are separated and developed on a two-dimensional plane. Suitable for acrylamide gel electrophoresis.

  The electrophoresis apparatus is not limited to the one shown here, and a part of the configuration may be changed as appropriate within a range not impeding the effects of the present invention. For example, except for the electrophoresis kit 10A, it can be configured in the same manner as a conventional apparatus.

An example of analysis conditions for analyzing a protein having a molecular weight in the range of about 10 to 200 kDa is shown below. In the following, the unit of concentration “M” represents “mol / L”.
(1) Transfer film 31: made of PVDF, X ₃ is 100 mm and Y ₃ is 55 mm (2) Protective sheet 32: PET film with low liquid permeability and thermal conductivity, and colored PET with low light permeability A plastic film laminated with a film having X ₄ of 15 mm and Y ₄ of 55 mm. (3) Driving auxiliary member 33: made of acrylic resin and having a strip shape of 55 mm × 200 mm. (4) First connecting portion 30a. : Connection width X ₃₁ is 5 mm and thermally welded (5) Second connection portion 30 b: Connection width X ₃₂ is 5 mm and heat welded (6) Gel 200: Separation of concentration 12.5% (7) Cathode buffer: Tris (25 mM) / Glycine (192 mM) / SDS (0.2%) buffer (40 ml)
(8) Anode buffer: Tris (150 mM) / methanol (10%) buffer (40 ml)
(9) Moving speed of the composite transfer film 3: 10 μm / second (10) Electrophoretic conditions: constant current 40 mA, 2 hours Under the above conditions, the transfer film 31 moves 72 mm until the end of the transfer of molecules, during which the molecular weight Is separated and transcribed, and substantially all the proteins contained in the sample can be separated and analyzed.
However, the conditions shown here are only examples of what can be applied, and it goes without saying that the conditions can be changed as appropriate according to the purpose.

  Up to this point, the case where the electrophoresis kit is used as the separation and adsorption apparatus and analysis method according to the present invention has been described. However, as the separation and adsorption apparatus, the separation and adsorption kit and a driving unit that moves the adsorption member, Can be applied in accordance with the molecule to be analyzed. Moreover, as an analysis method, if it has the process of adsorb | sucking the isolate | separated molecule | numerator to an adsorption | suction member from a separation medium, various structures can be applied according to a separation adsorption apparatus.

  The present invention can be used in all fields where analysis of macromolecules is required, such as medicine, pharmacy, biochemistry, and food.

  DESCRIPTION OF SYMBOLS 1 ... Electrophoresis kit, 2 ... Gel filling tank, 2a ... 1st opening part, 2b ... 2nd opening part, 200 ... Gel, 3 ... Composite transfer film 31 ... Transfer film, 32 ... Protective sheet, 4 ... Drive means, 10A ... Electrophoresis kit, 5 ... Liquid permeable sheet, 99a ... Cathode buffer, 99b ..Anode buffer solution, 100A ... electrophoresis device

Claims (8)

A separation medium that separates molecules to be analyzed by passing an electric current through a buffer solution; a separation tank that stores the separation medium; and a porous adsorption member that adsorbs the separated molecules from the separation medium. Separation adsorption kit,
The separation tank has first and second openings for flowing current,
The adsorbing member is provided with a protection unit that protects the exposed surface of the separation medium,
The separation / adsorption kit, wherein the adsorption member is arranged so that the protection portion is in close contact with an exposed surface of the separation medium in the second opening.   The separation / adsorption kit according to claim 1, wherein the protection unit is connected to the adsorption member.   The separation adsorption kit according to claim 1 or 2, wherein the protection part is provided on the adsorption member. The separation / adsorption kit according to claim 1 , wherein a part of the adsorbing member is changed into the protection unit.   The separation adsorption kit according to any one of claims 1 to 4, wherein the protection part is bonded to the second opening.   The separation / adsorption kit according to claim 1, wherein a conductive medium that allows the molecules to pass therethrough is provided between the second opening and the protection part. A separation and adsorption device comprising the separation and adsorption kit according to any one of claims 1 to 6,
Furthermore, the separation adsorption apparatus characterized by having a drive means for moving the adsorption member in the separation adsorption kit. A molecular analysis method using the separation and adsorption kit according to any one of claims 1 to 6,
A method for analyzing molecules, comprising: moving the adsorbing member, moving the protection unit from the second opening, and adsorbing the separated molecules from the separation medium to the adsorbing member.

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