JP2012088167A - Two dimensional electrophoretic cassette, and manufacturing method of two dimensional electrophoretic cassette - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent bubbles from remaining in a gel, and charge a gel precursor liquid efficiently.SOLUTION: In a two dimensional electrophoretic cassette, a first opening (opening) 11 penetrating through a first board (board) 2a and communicating a cavity C and a first buffer tank (buffer tank) 3; a second opening (opening) 12 penetrating through the first board (board) 2a and communicating the cavity C and the second buffer tank (buffer tank) 4; a charging hole 20 penetrating through a second board (board) 2b and allowing the gel precursor liquid Ga to be charged into the cavity C from outside the cassette body 10 are formed. The two dimensional electrophoretic cassette is provided with a first sealing material (sealing material) 31 capable of sealing the first opening 11 and attachable/detachable to/from the first board 2a; and a second sealing material (sealing material) 32 capable of sealing the second opening 12 and attachable/detachable to/from the first board 2a.

Description

  The present invention relates to a cassette for two-dimensional electrophoresis and a method for producing a cassette for two-dimensional electrophoresis.

  After the completion of the Human Genome Project, proteome research has been actively conducted. This “proteome” means the total of proteins produced by translation in specific cells, organs, and organs, and examples of research on the proteome include protein profiling.

  As one of the techniques for profiling proteins, two-dimensional electrophoresis of proteins is known. This two-dimensional electrophoresis is composed of two electrophoretic steps: isoelectric focusing that separates proteins depending on charge, and slab gel electrophoresis that separates proteins depending on molecular weight (especially SDS-PAGE). (For example, refer to Patent Document 1).

  Patent Document 2 discloses an example of a sample separation apparatus that performs the two-dimensional electrophoresis. The sample separation device includes a first chip that is separated by isoelectric focusing and a second chip that performs slab gel electrophoresis.

The first chip is composed of a support plate and a first gel (first-dimensional gel) supported on the support plate. In this first chip, a sample (sample) is introduced into the first gel, and after passing through the step of swelling the first gel, a voltage is applied to the first gel to separate the sample in the first direction. (1st dimension separation). Then, the first chip is transferred to the second chip after being subjected to the process of staining the separated sample and the process of equilibrating to the environment of the second chip.
The second chip is configured by storing a second gel (second-dimensional gel) in the substrate, and the first gel of the first chip is connected to the second gel. In this state, by applying a voltage to the second gel, the sample is separated in a second direction different from the first direction (two-dimensional separation).

  Patent Document 2 describes an electrophoresis cassette integrated with a buffer tank, in which a buffer tank for storing a buffer solution for an electrode for electrophoresis is provided in a chip. Unlike the conventional two-plate precast gel plate structure, the electrophoresis cassette is equipped with a buffer tank in the electrophoresis cassette itself, so a buffer tank is prepared separately from the electrophoresis cassette. There is no need to do this and it is convenient.

Japanese Patent Laid-Open No. 11-30605 JP 2007-64848 A

  In the electrophoresis cassette integrated with the buffer tank as described above, an opening for communicating the space in which the gel is accommodated with the buffer tank is formed so that the gel and the electrophoresis buffer can be brought into contact with each other. For this reason, when the electrophoresis precursor cassette is filled with the gel precursor solution, air from the opening easily enters the gel precursor solution, and bubbles may remain in the vicinity of the opening in the formed gel. And this bubble may become a defect which affects electrophoresis.

In addition, in the step of filling the gel precursor solution into the buffer tank-integrated electrophoresis cassette, the gel precursor solution is filled into the electrophoresis cassette from this opening, so the gel precursor solution is filled for each electrophoresis cassette. There is a problem in mass productivity and workability.
In addition, since the opening is sealed after the step of filling the gel cassette with the gel precursor solution, an operation such as fitting a sealing material into the opening is required, and there are problems in mass productivity and workability.
Further, the operation of inserting the sealing material may cause bubbles to remain in the gel formed by forming bubbles in the gel precursor solution.

  The present invention has been made in view of the above-described problems, and is capable of preventing bubbles from remaining in a gel and efficiently filling a gel precursor solution with a two-dimensional electrophoresis cassette and two-dimensional electricity. It aims at providing the manufacturing method of the cassette for electrophoresis.

  In order to achieve the above object, the two-dimensional electrophoresis cassette according to the present invention has a gel housed in a cavity portion of a cassette body configured by combining substrates, and a buffer tank is formed adjacent to the cassette body. A cassette for two-dimensional electrophoresis, which includes an opening that penetrates the substrate and allows the cavity and the buffer tank to communicate with each other, and the gel precursor is filled from the outside of the cassette body into the cavity through the substrate. And a filling material that is capable of sealing the opening and detachable from the substrate.

  In the present invention, an opening that allows the cavity and the buffer tank to communicate with each other through the substrate and a filling hole that penetrates the substrate and can be filled with the gel precursor solution from the outside of the cassette body are formed. When the gel precursor solution is filled in the cavity, the opening can be sealed and the gel precursor solution can be filled from the filling hole. Therefore, air does not enter the filled gel precursor solution from the opening, and bubbles can be prevented from remaining in the formed gel.

In the two-dimensional electrophoresis cassette according to the present invention, the opening may be formed in a tapered surface whose inner peripheral surface extends from the buffer tank side toward the cavity portion side.
In the two-dimensional electrophoresis cassette according to the present invention, it is preferable that the opening has an intersecting angle between the tapered surfaces of 90 ° or more in a cross-sectional view orthogonal to the surface direction of the substrate.
As described above, the opening is formed in a tapered surface whose inner peripheral surface is widened from the buffer tank side toward the cavity portion side, so that when the gel precursor solution is filled in the cavity portion, the opening portion has air. Therefore, it is possible to prevent bubbles from remaining in the formed gel.

In the two-dimensional electrophoresis cassette according to the present invention, the sealing material is preferably an adhesive sealing material or a pressure-sensitive crimping sealing material.
Thus, the opening can be reliably sealed by the sealing material being an adhesive sealing material or a pressure-sensitive pressure-bonding sealing material.

Further, in the two-dimensional electrophoresis cassette according to the present invention, the sealing material can be fitted into the opening, and when the fitting is fitted into the opening, the end surface on the cavity side is formed on the substrate. It may be flush with the inner peripheral surface.
As described above, the sealing material can be fitted into the opening, and when the end face on the cavity side is flush with the inner peripheral surface of the substrate when fitted into the opening, air is introduced into the opening. Can be prevented and air bubbles can be prevented from remaining in the formed gel.

In the two-dimensional electrophoresis cassette according to the present invention, it is preferable that the sealing material is made of silicon rubber or urethane rubber.
Thus, since the sealing material is formed from silicon rubber or urethane rubber, the opening can be reliably sealed.

  The method for manufacturing a cassette for two-dimensional electrophoresis according to the present invention includes a gel precursor solution filling step of filling a cavity portion of a cassette body configured by combining substrates with a gel precursor solution, and gelling the gel precursor solution. A two-dimensional electrophoresis cassette comprising a gelling step, wherein the two-dimensional electrophoresis cassette has a buffer tank formed adjacent to the cassette body, and penetrates the substrate to form the cavity portion. And an opening for communicating with the buffer tank, and a filling hole that penetrates the substrate and is capable of filling the cavity with the gel precursor from the outside of the cassette body, the gel precursor solution filling step Then, the substrate is arranged so that the filling hole is at the top, the opening is sealed, and the cavity portion is filled with the gel precursor solution from the filling hole. And wherein the Rukoto.

In the present invention, in the gel precursor solution filling step, the opening is sealed and the gel precursor solution is filled into the cavity from the filling hole, so that air does not enter the filled gel precursor solution from the opening. It is possible to prevent bubbles from remaining in the formed gel.
Compared to the conventional method of filling the cavity with the gel precursor solution from the opening, and then filling the gel precursor solution with the sealant, the gel precursor solution contains the gel precursor solution. Since air does not enter and an operation for attaching the sealing material can be performed in advance, a two-dimensional electrophoresis cassette can be efficiently manufactured.

In the method for manufacturing a cassette for two-dimensional electrophoresis according to the present invention, a plurality of the cassette bodies may be arranged side by side, and the gel precursor solution filling process may be simultaneously performed on the cavity portions of the plurality of cassette bodies.
As described above, by performing the gel precursor solution filling step simultaneously on the cavity portions of the plurality of cassette bodies, a large number of two-dimensional electrophoresis cassettes can be efficiently manufactured.

  According to the present invention, it is possible to prevent bubbles from remaining in the formed gel, to efficiently manufacture a two-dimensional electrophoresis cassette, and to improve the quality of the two-dimensional electrophoresis cassette. Can do.

It is a figure which shows an example of the cassette for two-dimensional electrophoresis by 1st Embodiment of this invention. It is the sectional view on the AA line of FIG. It is the figure which looked at the cassette for two-dimensional electrophoresis from the filling hole side. (A) The figure explaining the cassette for two-dimensional electrophoresis by 2nd Embodiment of this invention, (b) is a figure explaining the taper surface (inner peripheral surface) of a 1st opening part and a 2nd opening part. It is a figure explaining the manufacturing method of the cassette for two-dimensional electrophoresis of this invention. It is a figure explaining the cassette for two-dimensional electrophoresis by 3rd Embodiment of this invention. It is a figure explaining the cassette for two-dimensional electrophoresis by 4th Embodiment of this invention.

(First embodiment)
Hereinafter, a two-dimensional electrophoresis cassette according to a first embodiment of the present invention will be described with reference to FIGS.
A two-dimensional electrophoresis cassette 1A according to the present embodiment shown in FIGS. 1 to 3 is used for, for example, two-dimensional electrophoresis for analyzing a sample such as a protein, and contains a gel G for two-dimensional electrophoresis inside. Is filled.

As shown in FIGS. 1 to 3, the two-dimensional electrophoresis cassette 1A includes a cassette body 10 configured by combining a first substrate 2a and a second substrate 2b made of synthetic resin. The first substrate 2a and the second substrate 2b are integrated by adhesion or welding.
As shown in FIG. 2, a substantially rectangular parallelepiped cavity C is formed between the first substrate 2a and the second substrate 2b, and the gel G used for electrophoresis is contained in the cavity C. Is formed.
In the present embodiment, the migration direction in which the sample migrates in the gel G is the direction of the arrow A in FIGS.

As shown in FIGS. 1 and 2, the first substrate 2 a is provided with a first buffer tank 3 and a second buffer tank 4, and the first buffer tank 3 migrates a sample more than the second buffer tank 4. It is arranged on the rear side in the direction (arrow A).
The 1st buffer tank 3 and the 2nd buffer tank 4 are comprised by the container shape which can inject | pour the electrophoresis buffer (running buffer) used for electrophoresis. In addition, a pair of electrodes of a DC power supply (not shown) can be connected to each of the first buffer tank 3 and the second buffer tank 4. The sample can be electrophoresed in the gel G by applying a DC voltage to the gel G.

The first substrate 2 a is formed with a first opening 11 that allows the cavity C and the first buffer tank 3 to communicate with each other. The first opening 11 is formed in a slit shape penetrating the first substrate 2a.
Since the first opening 11 is formed in the first substrate 2a, the gel G can be energized through the running buffer supplied to the first buffer tank 3, and the connection with the first-dimensional gel can be established. It can be carried out.

A second opening 12 is formed in the first substrate 2a so that the cavity C and the second buffer tank 4 communicate with each other. The second opening 12 is formed in a slit shape penetrating the first substrate 2a.
By forming the second opening 12 in the first substrate 2a, the gel G can be energized through the running buffer supplied to the second buffer tank 4.

As for the 1st opening part 11 and the 2nd opening part 12, the internal peripheral surfaces 11a and 12a are orthogonally crossed to the surface direction of the 1st board | substrate 2a.
The first opening 11 and the second opening 12 are respectively sealed by the first sealing material 31 and the second sealing material 32 from the time of manufacture of the two-dimensional electrophoresis cassette 1A to before use. . This prevents the gel precursor liquid Ga, which is a precursor of the gel G, from flowing out of the first opening 11 and the second opening 12 during the manufacture of the two-dimensional electrophoresis cassette 1A, and is two-dimensional. Drying of the gel G is suppressed during the period from the manufacture of the electrophoresis cassette 1A to the use of the two-dimensional electrophoresis cassette 1A.
When the two-dimensional electrophoresis cassette 1A is used, the first opening 11 and the second opening 12 are in an opened state with the sealing materials 31 and 32 removed.
Here, the sealing material that seals the first opening 11 will be described as a first sealing material 31, and the sealing material that seals the second opening 12 will be described as a second sealing material 32.

The first sealing material 31 is detachable from the first substrate 2a, for example, an adhesive sealing material, a pressure-sensitive pressure-bonding sealing material, etc., and is large enough to cover the first opening 11 from the first buffer tank 3 side. Is formed.
The second sealing material 32 can also be attached to and detached from the first substrate 2a. For example, the second opening 12 can be covered from the second buffer tank 4 side with an adhesive sealing material, a pressure-sensitive pressure-bonding sealing material, or the like. It is formed in a large size.

In the second substrate 2b, a plurality of filling holes 20 are formed at the rear end in the sample migration direction (arrow A) to communicate the cavity C with the outside of the second substrate 2b.
The filling hole 20 is formed so that a filling nozzle (not shown) for filling the cavity portion C with the gel precursor solution Ga is inserted when the two-dimensional electrophoresis cassette 1A is manufactured.
In the present embodiment, a plurality of filling holes 20 are formed in the second substrate 2b, but a single filling hole 20 may be used.

Next, a method for manufacturing the above-described two-dimensional electrophoresis cassette 1A will be described.
First, as shown in FIG. 2, the cavity portion C is configured by combining the first substrate 2 a and the second substrate 2 b formed into a predetermined shape.
At this time, the first opening 11 is covered and sealed with the first sealing material 31, and the second opening 12 is covered and sealed with the second sealing material 32.

Subsequently, the cavity portion C is filled with a gel precursor solution Ga having a predetermined concentration (gel precursor solution filling step filling step).
In the gel precursor solution filling step, as shown in FIG. 2, the cassette body 10 is positioned so that the front side in the sample migration direction (arrow A) is directed toward the gravitational direction (lower side), and the filling hole 20 is on the upper side. Install in.

Then, the cavity portion C is filled with the gel precursor solution Ga from a filling nozzle (not shown).
Here, an acrylamide gel is mainly used as the gel G filled in the cavity portion C. As the acrylamide gel preparation solution (gel precursor solution Ga), those described in the well-known electrophoresis method manual can be used, and there is no particular limitation, but crosslinking of acrylamide monomers, N, N-methylenebisacrylamide, etc. It is charged after adding a polymerization initiator to a monomer solution comprising an agent, a pH buffering agent, and if necessary, a modifying agent such as SDS.

  At this time, the gel precursor solution Ga may be continuously filled with a uniform concentration, or may be filled while changing the concentration. In order to create a polyacrylamide gel having a concentration gradient, a known method of filling separately prepared high-concentration acrylamide solution and low-concentration acrylamide solution while changing the mixing ratio with time can be used. At this time, a high specific gravity substance such as glycerin may be added to one of the two types of solutions to give a specific gravity difference between the solutions.

Subsequently, the gel precursor solution Ga filled in the cavity portion C is gelled (gelation step).
After filling the cavity portion C with the gel precursor solution Ga, the filling hole 20 is sealed with a film or the like, and the cassette body 10 filled with the gel precursor solution Ga in the cavity portion C is allowed to stand for a sufficient time until polymerization proceeds. Let
Then, the gel precursor solution Ga is gelled, whereby the two-dimensional electrophoresis cassette 1A is manufactured.

Next, effects of the above-described two-dimensional electrophoresis cassette 1A will be described with reference to the drawings.
According to the two-dimensional electrophoresis cassette 1A according to the present embodiment, the first sealing material 31 that can seal the first opening 11 and the second sealing material 32 that can seal the second opening 12 are provided. The first opening 11 and the second opening 12 are sealed in the step of filling the gel precursor liquid Ga into the cavity C by forming the filling hole 20 for filling the gel precursor liquid Ga. Thus, the gel precursor solution Ga can be filled from the filling hole 20.
For this reason, air does not enter the filled gel precursor solution Ga from the first opening 11 and the second opening 12, and bubbles can be prevented from remaining in the formed gel G. In addition, the quality of the two-dimensional electrophoresis cassette 1A can be efficiently manufactured.

(Second Embodiment)
Next, other embodiments will be described with reference to the accompanying drawings. However, the same or similar members and parts as those of the above-described embodiments are denoted by the same reference numerals, and the description thereof is omitted. The configuration is different from that of the first embodiment. Will be described.
As shown in FIG. 4A, in the two-dimensional electrophoresis cassette 1B according to the second embodiment, the inner peripheral surface 13a of the first opening 13 and the inner peripheral surface 14a of the second opening 14 are the first substrate. It is formed in the taper surface which spreads outward from the 1st buffer tank 3 and 2nd buffer tank 4 side of 2a toward the cavity part C side.

According to the two-dimensional electrophoresis cassette 1B according to the second embodiment, since the inner peripheral surface 13a of the first opening 13 and the inner peripheral surface 14a of the second opening 14 are formed as tapered surfaces, the inside of the cavity C When the gel precursor solution Ga is filled in, air does not easily accumulate in the first opening 13 and the second opening 14, so that bubbles can be prevented from remaining in the formed gel G.
In particular, as shown in FIG. 4B, the crossing angle θ between the tapered surfaces (inner peripheral surfaces 13a, 14a) in a cross-sectional view orthogonal to the surface direction of the first substrate 2a is 90 ° or more. Further, no residual gas bubbles are observed in the gel G, which is more preferable.

Further, in the method for manufacturing the two-dimensional electrophoresis cassette 1B according to the second embodiment, a plurality of cassette bodies 10 are arranged side by side as shown in FIG. 5, and the gel precursor solution is supplied from the filling holes 20 of the plurality of cassette bodies 10 to the cavity portion C. Ga is filled at the same time.
Thus, the two-dimensional electrophoresis cassette 1B can be efficiently manufactured by simultaneously filling the plurality of cassette bodies 10 with the gel precursor solution Ga.

(Third embodiment)
As shown in FIG. 6, in the two-dimensional electrophoresis cassette 1 </ b> C according to the third embodiment, the first sealing material 33 is fitted into the first opening 11, and the second sealing material 34 is connected to the second opening 12. Is fitted.
The first sealing material 33 includes a convex portion 33 a corresponding to the first opening 11. The height of the convex portion 33a is substantially equal to the thickness of the first substrate 2a, and the tip end surface 33b is substantially flush with the surface of the first substrate 2a on the cavity portion C side.
Similar to the first sealing material 33, the second sealing material 34 also includes a convex portion 34 a corresponding to the second opening 12. The height of the convex portion 34a is substantially equal to the thickness of the first substrate 2a, and the tip end surface 34b is substantially flush with the surface of the first substrate 2a on the cavity portion C side.
As the first sealing material 33 and the second sealing material 34, an adhesive material or the like is prepared at a portion that is in close contact with the cassette by a resin molding, or molding of silicon rubber or urethane rubber that can be in close contact with the cassette Things are preferred.

According to the two-dimensional electrophoresis cassette 1 </ b> C according to the third embodiment, the first sealing material 33 and the second sealing material 34 are formed with convex portions 33 a and 34 a, and the convex portion 33 a is the first opening 11. Further, the first opening portion 11 and the second opening portion are fitted by fitting the convex portion 34a into the second opening portion 12 and having the tip end surfaces 33b and 34b substantially flush with the inner peripheral surface of the first substrate 2a. Air does not accumulate in the portion 12, and bubbles can be prevented from remaining in the formed gel G.
In addition, the first sealing material 33 and the second sealing material 34 are made of a resin molded product in which an adhesive material or the like is prepared in close contact with the cassette, or silicon rubber or urethane rubber that can be in close contact with the cassette. By being a molding, the 1st opening part 11 and the 2nd opening part 12 can be sealed, and the inflow of the air from the 1st opening part 11 and the 2nd opening part 12 can be prevented.

(Fourth embodiment)
As shown in FIG. 7, in the two-dimensional electrophoresis cassette 1D according to the fourth embodiment, the first substrate 53a is a plate member 53c that partitions the cavity C from the first buffer tank 3 and the second buffer tank 4. It is formed thinner than the plate member 2 c that partitions the cavity C of the first embodiment shown in FIG. 2 and the first buffer tank 3 and the second buffer tank 4. Therefore, the depths of the first opening 51 and the second opening 52 are formed shallower than the depths of the first opening 11 and the second opening 12 of the first embodiment shown in FIG.
Further, the plate member 52c of the first substrate 52b is formed to be thinner than the second substrate 53b.
The two-dimensional electrophoresis cassette 1D according to the fourth embodiment has less air remaining in the first opening 51 and the second opening 52 than the two-dimensional electrophoresis cassette 1A according to the first embodiment. Since the air in the 1st opening part 51 and the 2nd opening part 52 is easy to be discharged | emitted, it can prevent that a bubble remains in the formed gel G more.

The embodiment of the two-dimensional electrophoresis cassette according to the present invention has been described above. However, the present invention is not limited to the above-described embodiment, and can be changed as appropriate without departing from the spirit of the present invention.
For example, in the method for manufacturing a cassette for two-dimensional electrophoresis according to the second embodiment, the cassette body 10 is filled with the gel precursor solution Ga at the same time, but the cassette body 10 is filled with the gel precursor solution Ga one by one. Good. In the method for manufacturing a two-dimensional electrophoresis cassette according to the first embodiment, a plurality of cassette bodies 10 may be arranged side by side, and the plurality of cassette bodies 10 may be simultaneously filled with the gel precursor solution Ga.
In the above embodiment, the first substrate 2a and the second substrate 2b are made of synthetic resin, but may be made of glass.

1A to 1D 2D electrophoresis cassette 2a, 53a First substrate (substrate)
2b, 53b Second substrate (substrate)
3 1st buffer tank 4 2nd buffer tank 10 Cassette main body 11,13,51 1st opening part 12,14,52 2nd opening part 11a, 12a, 13a, 14a Inner peripheral surface 20 Filling hole 31,33 1st sealing Stop material (encapsulant)
32, 34 Second sealing material (sealing material)
C cavity part G gel Ga gel precursor θ crossing angle

Claims (8)

A two-dimensional electrophoresis cassette in which a gel is accommodated in a cavity portion of a cassette body configured by combining substrates, and a buffer tank is formed adjacent to the cassette body,
An opening that penetrates the substrate and communicates the cavity and the buffer tank;
A filling hole that penetrates the substrate and can be filled with the gel precursor solution from the outside of the cassette body to the cavity portion is formed;
A cassette for two-dimensional electrophoresis, comprising a sealing material capable of sealing the opening and detachable from the substrate.   2. The two-dimensional electrophoresis cassette according to claim 1, wherein the opening is formed in a tapered surface whose inner peripheral surface extends from the buffer tank side toward the cavity portion side.   The cassette for two-dimensional electrophoresis according to claim 2, wherein the opening has an intersecting angle of 90 ° or more between tapered surfaces in a cross-sectional view orthogonal to the surface direction of the substrate.   4. The two-dimensional electrophoresis cassette according to claim 1, wherein the sealing material is an adhesive sealing material or a pressure-sensitive pressure-bonding sealing material. 5.   The sealing material can be fitted into the opening, and an end surface on the cavity side is flush with an inner peripheral surface of the substrate when fitted into the opening. Item 2. The two-dimensional electrophoresis cassette according to Item 1.   6. The two-dimensional electrophoresis cassette according to claim 5, wherein the sealing material is made of silicon rubber or urethane rubber. A gel precursor solution filling step of filling a gel precursor solution into a cavity portion of a cassette body configured by combining substrates;
A gelling step of gelling the gel precursor solution, and a method for producing a cassette for two-dimensional electrophoresis comprising:
The two-dimensional electrophoresis cassette has a buffer tank formed adjacent to the cassette body, and has an opening penetrating the substrate and communicating the cavity and the buffer tank, penetrating the substrate and the cassette body. A filling hole capable of filling the gel precursor solution into the cavity portion from the outside is further formed,
In the gel precursor solution filling step, the substrate is arranged so that the filling hole is on the upper side, the opening is sealed, and the cavity is filled from the filling hole with the gel precursor solution. A method for producing a cassette for two-dimensional electrophoresis. The method for producing a cassette for two-dimensional electrophoresis according to claim 7, wherein a plurality of the cassette bodies are arranged side by side, and a gel precursor solution filling step is simultaneously performed in the cavity portions of the plurality of cassette bodies.

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