JP4586179B2 - Sample injection instrument for two-dimensional electrophoresis, two-dimensional electrophoresis apparatus including the same, and two-dimensional electrophoresis using the apparatus - Google Patents

Description

  The present invention relates to a sample injection instrument for two-dimensional electrophoresis, a two-dimensional electrophoresis apparatus including the same, and a two-dimensional electrophoresis method using the apparatus.

  Two-dimensional electrophoresis is a technique widely used for detection and separation of components in a sample in fields such as chemistry, biochemistry, and molecular biology. In many cases, isoelectric focusing is performed using an elongated strip-shaped first-dimensional gel as a medium, and then the first-dimensional gel is moved to another apparatus to perform second-dimensional electrophoresis. In recent years, the gel for the first dimension and the gel for the second dimension are placed by separating the gel for the first dimension and the gel for the second dimension on a single support substrate and filling the gap between them. A method of performing two-dimensional electrophoresis simply by connecting gels with a gel and performing second-dimensional electrophoresis in a direction perpendicular to the first dimension has been performed (Patent Document 1). However, a threshold is required to uniformly add the sample to the entire first-dimensional gel and prevent it from flowing out to other parts, and it is necessary to remove the threshold before the first-dimensional electrophoresis.

  In order to perform two-dimensional electrophoresis accurately, that is, to move components in the sample to the correct migration position, it is desirable to add the sample as uniformly as possible to the first-dimensional gel. When a sample is manually added, it is complicated and skillful to add a small amount of sample uniformly to the entire gel.

  Conventionally, as an instrument or apparatus for adding a sample to an electrophoresis gel or the like, for example, an electrophoresis chip having advantages such as improving the degree of freedom of a liquid reservoir and performing more analysis (patent) Reference 2), sample injection device (U.S. Pat. No. 6,099,097) equipped with a urea removal unit that removes urea eluted from the gel, a small amount that can reliably inject the absolute sample amount and visually confirm that the sample injection is complete A sample injector (Patent Document 4) and the like are known. However, there is no known sample injection device or apparatus having a configuration for the purpose of uniformly and simply adding a sample to the first-dimensional gel of the two-dimensional electrophoresis method.

Japanese Patent No.27019443 Japanese Patent Laid-Open No. 2003-4700 Japanese Patent Laid-Open No. 3-2557 JP 2000-186985 A

  The object of the present invention is to provide a two-dimensional electrophoretic method in which a sample can be easily and uniformly added to a first-dimensional gel in a two-dimensional electrophoresis method, and moreover, two-dimensional electrophoresis can be easily performed on one support substrate. To provide an electrophoresis apparatus, a sample injection instrument used therefor, and a two-dimensional electrophoresis method using the same.

  As a result of earnest research, the inventors of the present application have used a sample injection device in which a first-dimensional dry gel is arranged so as to block the bottom of the slot-shaped sample injection port subjected to hydrophilic treatment on the inner wall. By injecting the sample from the sample injection port, the sample can be uniformly and easily added to the first-dimensional gel, and the first-dimensional gel and the second-dimensional gel are connected by injecting the gel without removing the threshold. Then, after conceiving a method of performing the second-dimensional electrophoresis, the present invention was completed.

  That is, the present invention provides a plate-shaped injection device main body, a sample injection port in a slot shape provided in the injector main body, the sample injection port whose inner wall is hydrophilized, Provided is a sample injection device for two-dimensional electrophoresis comprising a dry gel for first-dimensional electrophoresis arranged at the bottom. The present invention further includes an apparatus main body, a second-dimensional electrophoresis gel supported on the apparatus main body, and the sample injection device of the present invention disposed on the apparatus main body. Provided is a two-dimensional electrophoresis apparatus in which a gap as a junction portion is formed between an eye electrophoresis gel and the second-dimensional electrophoresis gel. Furthermore, the present invention includes a step of injecting a sample into the sample injection port of the sample injection device of the apparatus for two-dimensional electrophoresis of the present invention, a step of performing a first-dimensional electrophoresis, and then a junction portion. Provided is a two-dimensional electrophoresis method including a step of filling a gel and then performing a second-dimensional electrophoresis.

  According to the present invention, in a two-dimensional electrophoresis method, a two-dimensional electrophoresis apparatus capable of easily and uniformly adding a sample to a first-dimensional gel, a sample injection instrument used therefor, and a two-dimensional electrophoresis using the same The law was provided for the first time. In the sample injection device for two-dimensional electrophoresis of the present invention, the inner wall of the slot-like sample injection port is subjected to a hydrophilic treatment, and the first-dimensional gel is in a dry state so as to block the bottom of the sample injection port. Therefore, when the sample is injected into the sample injection port, the sample quickly spreads over the entire first-dimensional gel. Moreover, since the first-dimensional gel is fixed to the bottom of the sample inlet in advance, there is no need for operations such as alignment between the inlet and the gel, and the sample is simply injected into the slot-like sample inlet. Therefore, the operation is very simple because the sample quickly reaches the entire first dimension gel. Further, the sample does not flow out to a portion other than the first-dimensional gel, and can be connected by filling the gel with the first-dimensional gel and the second-dimensional gel without removing the limit. Therefore, by using the sample injection device of the present invention, the workability of two-dimensional electrophoresis is improved and the accuracy of two-dimensional electrophoresis is improved because the sample can be uniformly added to the first-dimensional gel. To do.

  Hereinafter, preferred specific examples of the present invention will be described with reference to the drawings.

FIG. 1 is a perspective view showing a preferred specific example of the two-dimensional electrophoresis apparatus of the present invention. FIG. 2 shows a cross-sectional structure by removing one side surface of the apparatus shown in FIG. It is a perspective view which shows the structure of the bottom part by inverting an injection device upside down. 1 and 2, the sample injection device 10 includes a flat injection device main body 12, and the injection device main body 12 is provided with a sample injection port 14. The sample inlet 14 has a slot shape, that is, an elongated through hole as shown. The width of the sample inlet 14 (the length in the direction perpendicular to the longitudinal direction) is not particularly limited, but is preferably 0.3 mm to 1.0 mm, and more preferably 0.3 mm to 0.7 mm. The length (length in the longitudinal direction) of the sample injection port 14 is not particularly limited, and is usually about 30 mm to 300 mm. A dry gel 16 for one-dimensional electrophoresis (FIG. 2) is disposed at the bottom of the sample inlet 14, and the bottom of the sample inlet 14 is closed by the dry gel 16. The size of the dry gel 16 is not particularly limited, and a size that protrudes from each side of the injection port 14 by about 0.1 mm to 3.0 mm is preferable. The thickness of the dried gel 16 is not particularly limited, but is usually about 0.01 mm to 0.1 mm (the thickness after swelling is usually about 0.3 mm to 1 mm). In addition, as the dry gel 16, what is used as the first-dimensional gel of the conventional two-dimensional electrophoresis such as agarose or polyacrylamide can be used as it is, and in many cases, the isoelectric point having a pH gradient is used. It is a gel for electrophoresis. The entire bottom surface of the sample inlet 14 is preferably closed with a dry gel 16. A pair of electrodes 18, 18 ′ for applying a voltage to the gel 16 are arranged at both ends of the dry gel 16 for first-dimensional electrophoresis. Further, the electrodes 18 and 18 ′ are not necessarily in contact with the gel 16 in a dry state, and may be disposed at a position in contact with the swollen first-dimensional gel 16 during operation. 1 and 2, reference numeral 20 denotes a two-dimensional electrophoresis apparatus main body, and the structure of the apparatus main body 20 will be described later.

The structure of the sample injection device 10 will be described in more detail with reference to FIGS. 3A, 3B, and 3C are a plan view, a bottom view, and a front view of the sample injection device 10 , respectively. Of the injection device main body 12, a region 12 a (hereinafter referred to as “junction portion of the junction portion”) that covers a junction portion (a gap between the first-dimensional gel and the second-dimensional gel, 40 in FIG. 2) of the electrophoresis apparatus described later. The lid is sometimes referred to as a “lid”, and is provided with a gel liquefied material filling hole 19 that is a through hole for filling the junction with the gel liquefied material. In the illustrated example, the electrodes 18, 18 ′ are disposed obliquely from the upper surface of the injector body 12, and the tip portions thereof are in contact with both end portions of the dry gel 16. The electrodes 18, 18 ′ may be in contact with the dried gel 16 or the swollen gel. For example, the electrodes 18, 18 ′ may be fixed to the lower surface of the injector main body 12 or disposed at both ends of the inlet 14. Is possible. The dry gel 16 is preferably supported on a support 22 as shown in FIG. The support is, for example, a plastic plate or film, and is preferably a plate. In this case, the dry gel 16 can be disposed at the bottom of the inlet 14 by suspending the support 22 on the lower surface of the injector body 12 with a pair of adhesive tapes 24, 24 ', as shown in the figure. it can.

The inner wall of the inlet 14 is hydrophilized. The hydrophilic treatment can be performed, for example, by coating a hydrophilic polymer, and this hydrophilic coating layer is indicated by reference numeral 26 in FIGS. 3 (A) and 3 (B). The hydrophilization treatment is performed in a range where the first-dimensional gel on the inner wall of the inlet 14 contacts. The method of hydrophilization treatment is not particularly limited, but the hydrophilic coating layer preferably has a uniform thickness in order to spread the sample uniformly throughout the gel, and the width of the inlet 14 is usually 1 mm. Since it is narrow as described below, it is not easy to apply the hydrophilic polymer with various known coaters. The inventors of the present application polymerize monomers in situ by making full use of plasma polymerization, and further treat with oxygen plasma or the like as necessary, so that the inner wall of the narrow inlet 14 has a thin uniform layer thickness. It has been found that it can be coated with a coating layer of a hydrophilic polymer. Monomers that can be used for plasma polymerization include monomers having a hydrophilic main chain such as ether, and monomers having a hydrophilic group such as a hydroxyl group, amino group, and carboxyl group in the main chain or side chain, The polymer is not particularly limited as long as it can be polymerized by plasma polymerization. Examples of preferable monomers include oxygen-containing organic compounds such as acrylic acid and propargyl alcohol, acetonitrile, aminoacetaldehyde dimethyl acetal, propylamine, allylamine, Examples include nitrogen-containing organic compounds such as pyridine, but are not limited thereto. Alternatively, hydrophilic treatment may be performed by plasma treatment after plasma polymerization of a hydrophobic layer such as hexamethyldisiloxane. The hydrophilic layer can also be formed by treating the inner wall itself with oxygen plasma or the like. That is, the hydrophilic layer is preferably
(A) treatment with at least one plasma selected from the group consisting of hydrogen plasma, oxygen plasma and nitrogen plasma;
(B) The coating layer formed by plasma polymerization of at least one selected from the group consisting of hexamethyldisiloxane and hexadiene is subjected to the plasma treatment of (a) above, or (c) acrylic acid, propargyl alcohol, acetonitrile, It can be formed by plasma polymerizing at least one selected from the group consisting of aminoacetaldehyde dimethyl acetal, propylamine, allylamine, and pyridine.

  The layer thickness of the hydrophilic layer is not particularly limited, but is preferably about 50 nm to 200 nm. The methods of plasma polymerization and plasma treatment are well known, and equipment for that is also commercially available. Therefore, it can be easily carried out using commercially available equipment, and the conditions for plasma polymerization and plasma treatment are also described in the following examples. It is specifically described. In addition, to increase the affinity with the gel filled in the junction after the first-dimensional electrophoresis, to ensure that the first-dimensional gel and the second-dimensional gel are connected uniformly throughout It is preferable to apply a hydrophilic treatment to the lower surface of the lid 12a. The hydrophilic treatment is preferably performed only on the inner wall of the inlet 14 and the lower surface of the lid 12a of the junction portion, and is not performed on other portions. By doing so, it is possible to prevent the sample liquid and the swelling liquid from leaking out to an unintended portion. For example, the hydrophilic treatment can be selectively performed only on the region to be subjected to the hydrophilic treatment by, for example, covering the region not subjected to the hydrophilic treatment with an adhesive film or the like and then performing a plasma polymerization treatment on the entire injection device.

  FIG. 4 is a cross-sectional view of the injection port 12 portion. FIG. 4A shows a state where the gel 16 is dried before use, and FIG. 4B shows a state after the gel 16 swells during electrophoresis. 4A and 4B are cross-sectional views of the vicinity of the injection port 14 as viewed from the side, and the right-hand drawings are cross-sectional views of the injection device as viewed from the front. . The size in FIG. 4 is an example of a preferred size, and is the size employed in the sample injection device produced in the following example. As shown in FIG. 4 (A), when the sample liquid 28 is injected into the sample injection port, the dried gel 16 comes into contact with the sample liquid 28 and swells, and its thickness increases as shown in FIG. 4 (B). Thus, the gel 16 ′ after swelling is obtained. The pressure-sensitive adhesive tapes 24, 24 'for suspending the support 22 of the dry gel 16 are preferably stretchable, which can extend according to the swelling of the dry gel 16, and the material thereof is, for example, rubber Vinyl, plastic and the like can be exemplified. Alternatively, a woven fabric, a non-woven fabric, or the like such as a commercially available adhesive bandage having elasticity can also be used.

Next, a two-dimensional electrophoresis apparatus will be described with reference to FIGS. The apparatus main body 20 includes a support base 30, and a second-dimensional gel 32 is supported on the support base 30. The support base 30 is surrounded by a frame body 34. The support base 30 and the frame body 34 are preferably formed integrally. Between both ends of the frame 34 in one direction and the support 30, buffer solution tanks 36 and 38 each having a groove formed by the absence of the support 30 are provided. In addition, on the one end region of the support base 30, the second-dimensional gel 32 does not exist, and a gap called the junction portion 40 exists. The junction portion 40 is a region that spatially separates the first-dimensional gel 16 and the second-dimensional gel 32 when the sample injection device 10 is placed on the frame 34 of the apparatus main body 20 . As the second-dimensional gel 32, a second-dimensional gel made of agarose, polyacrylamide or the like conventionally used for two-dimensional electrophoresis can be used as it is. The width (the left-right direction in FIG. 2) of the second-dimensional gel 32 is not particularly limited, and is usually about 40 mm to 300 mm. Moreover, it is preferable that length and thickness are comparable as the above-described first-dimensional gel 16. The material of the apparatus main body is not particularly limited, but an insulating material such as plastic is preferable. In addition, in the apparatus shown in FIG.1 and FIG.2, although the apparatus main body 20 and the sample injection instrument 10 are isolate | separated, you may form these integrally.

When performing two-dimensional electrophoresis, first, the sample injection device 10 is placed on the frame 34 so that the opening of the sample injection port 14 is on the upper side. Thereby, the first-dimensional dry gel 16 is disposed in the junction section 40. Further, the side surface on the frame side in the longitudinal direction of the first-dimensional dried gel 16 is separated from the frame body 34 by a gap and is not in contact with it, and the side surface on the second dimension gel 32 side is the junction portion 40. It is not in contact with the first dimension gel.

  Next, a sample solution is added to the sample inlet 14. Usually, the sample solution includes a swelling solution (including a protein denaturant, a surfactant, and the like). The swelling liquid is commonly used in conventional two-dimensional electrophoresis, and examples of preferred compositions are also described in the following examples. Also, the sample solution may be an arbitrary one containing components to be detected or separated by two-dimensional electrophoresis, as usual, and usually contains a biopolymer such as protein, sugar or nucleic acid. The sample solution is preferably added to the vicinity of the center of the sample inlet 14 in order to apply the sample as uniformly as possible to the entire gel. When the sample liquid is added to the inlet 14, the inner wall of the inlet 14 is hydrophilized as described above, so that the sample liquid instantly becomes a slot-shaped inlet despite the narrow width of the inlet. It spreads to both ends and is uniformly applied to the entire dried gel 16. The dried gel 16 swells when it comes into contact with the sample solution. After the gel 16 swells, electrophoresis is performed by applying a voltage to the electrodes 18, 18 '. The conditions such as voltage and migration time at this time are the same as in the prior art.

  After the first-dimensional electrophoresis as described above, a gel melt is injected from the gel liquefied material filling hole 19 and solidified to fill the junction portion 40 with the gel. The gel may be an agarose gel, polyacrylamide gel or the like, similar to the first-dimensional gel and the second-dimensional gel. In a preferred embodiment of the present invention, the portion that comes into contact with the gel is hydrophilized, and the bottom surface of the lid 12a of the junction portion is also hydrophilized as described above. The entire junction portion 40 is filled. After the filled gel is solidified, electrophoresis is performed in a direction orthogonal to the longitudinal direction of the first-dimensional gel using the second-dimensional gel as a medium. In this state, a buffer solution for second-dimensional electrophoresis is put in the buffer solution tanks 36 and 38, respectively, and a pair of electrodes (not shown) are inserted into the buffer solution in the buffer solution tanks 36 and 38. This can be done by applying a voltage between these electrodes. Note that conditions such as voltage and electrophoresis time in the second-dimensional electrophoresis are the same as those in the prior art.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

A sample injection device according to a preferred embodiment of the present invention shown in FIGS. 1 to 4 was produced. The sample inlet 14 was 0.5 mm wide and 52 mm long. Hydrophilic treatment was performed on the inner wall of the sample inlet 14 and the lower surface of the lid of the junction portion. Specifically, the hydrophilization treatment was performed as follows. That is, the portion of the injection device that was not subjected to the hydrophilic treatment was covered with a heat-resistant tape (Kapton tape, manufactured by Permacel), and then the injection device was placed in the bell jar of a plasma polymerization apparatus (Nihon Seed). After lowering the internal pressure in the bell jar to 1 × 10 ⁻³ Pa or less, first, oxygen plasma treatment was performed so that a hexamethyldisiloxane (HMDS) layer could be formed on the hydrophilic treatment surface. The treatment conditions were: RF power: 150 W, mass flow: 20 ml / min, treatment time: 180 seconds, and oxygen plasma treatment of the injection device. Thereafter, an HMDS layer having a thickness of about 100 nm was formed. The HMDS layer was layered under the conditions of layering: RF power: 150 W, mass flow: 100 ml / min, and processing time: 180 seconds. Finally, the layered poly HMDS layer was treated with oxygen plasma to form a hydrophilic layer. The oxygen plasma treatment conditions were 150 W, mass flow: 20 ml / min, treatment time: 60 seconds.

As the first-dimensional gel 16, a polyacrylamide dry pH gradient-immobilized gel (Invitrogen, thickness 0.02 mm × width in the short direction 0.8 mm × length in the longitudinal direction 52 mm) and capron at both ends in the longitudinal direction are used. It was fixed to the lower surface of the injection device by fastening with tape. At that time, the entire surface of the bottom of the sample injection port was closed with the dry gel 16. In addition, the upper surface of the dried gel 16 was attached to the lower surface of the injection device body, and the support plate 22 was directed in the opposite direction to the injection device body 12. The injection device 10 produced in this way was placed on one end on the frame 34 of the plastic device main body 20 . One side surface in the longitudinal direction of the dried gel 16 is blocked by a space so as not to contact the wall of the frame body 34. Further, the other side surface in the longitudinal direction of the dried gel 16 is separated from the second-dimensional gel 32 by the junction portion 40. The second-dimensional electrophoresis gel 32 was a polyacrylamide gel, and 375 mM Tris-HCl buffer (pH 8.8) was used as the buffer. As is clear from the above configuration, the first-dimensional gel 16 and the second-dimensional gel 32 are electrically insulated from each other.

  Next, protein denaturant (urea 6M, thiourea 2M), surfactant (CHAPS (manufactured by Dojindo)) 2% (w / v), reducing agent (dithiothreitol) 20 mM, carrier ampholite (manufactured by Invitrogen) A swelling solution containing 0.5% (v / v) was prepared, the swelling solution was added to the sample solution, and 20 μL of the sample solution containing the swelling solution was prepared. As a sample, a marker protein for two-dimensional electrophoresis (amyloglucosidase, ovalbumin, carbonic anhydrase, myoglobin) (manufactured by Sigma) was used. The protein was fluorescently labeled with Cy5 (Amersham Biosciences).

  The sample solution containing the sample-containing swelling solution was added to the injection device to swell the gel 1. After standing for 15 minutes from the addition, isoelectric focusing was performed using an electrode for first-dimensional electrophoresis.

Apply electric power within 60mW / mm ³ between the electrodes (0-5000 V (Linear) for 4 minutes, 5000-6000 V (Linear) for 1 minute, 6000 V for 5 minutes), and let electricity flow for a total of 10 minutes. did. At this time, in order to prevent drying and burning of the gel due to the generation of heat, the gel 16 was cooled by a Peltier device so as to be 0 to 5 ° C.

  125 mM Tris-HCl buffer solution (pH 6.8) and 0.25% agarose mixed at 95 ° C. through a gel liquefied material filling hole 19 provided in the lid 12a of the junction part of the injection device body 12 The liquid was injected. The agarose gelled within 5 minutes. Next, a buffer solution for second-dimensional electrophoresis (containing 0.19 M glycine, SDS, and 25 mM trisaminomethane) is injected into the buffer solution tanks 36 and 38, and the buffer solution tank 36 serves as the cathode and the buffer solution serves as the anode. The electrode was brought into contact with the tank 38, and second-dimensional electrophoresis was performed (constant voltage 200V, about 15 minutes).

  FIG. 5 shows an image of a second-dimensional polyacrylamide gel in which the sample is separated after the second-dimensional electrophoresis performed following the first-dimensional electrophoresis. As shown in FIG. 5, as a result of performing two-dimensional electrophoresis using the two-dimensional electrophoresis apparatus, each protein in the sample was well separated.

It is a perspective view of one preferable example of the two-dimensional electrophoresis apparatus of the present invention. FIG. 2 is a perspective view showing the two-dimensional electrophoresis apparatus shown in FIG. 1 with a part of the apparatus main body removed. FIG. 3 is a plan view, a bottom view, and a front view in the vicinity of a sample injection port of a preferred specific example of the sample injection device of the present invention shown in FIGS. 1 and 2. It is sectional drawing of the sample injection port vicinity of one preferable specific example of the sample injection device of this invention shown in FIG.1 and FIG.2. It is an electrophoresis photograph which shows the result of the two-dimensional electrophoresis performed in the Example of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Sample injection device 12 Injection device main body 14 Sample injection port 16 First-dimensional dry gel 18, 18 'Electrode for one-dimensional electrophoresis 19 Gel liquid filling hole
20 Two-dimensional Electrophoresis Device Body 22 Support 24, 24 ′ Adhesive Tape 26 Hydrophilic Coating Layer 28 Sample Solution 30 Support Base 32 Second Dimensional Gel 34 Frame 36 Buffer Solution Tank 38 Buffer Solution Tank 40 Junction Portion

Claims (12)

  A plate-shaped injection device main body, a slot-shaped sample injection port provided in the injector main body, the inner wall of which is hydrophilized, and a bottom of the sample injection port A sample injection instrument for two-dimensional electrophoresis comprising a dry gel for first-dimensional electrophoresis.   2. The sample injection according to claim 1, wherein the width of the sample injection port is 0.3 mm to 1.0 mm, and the hydrophilic treatment is performed by forming a hydrophilic layer on the inner wall by plasma treatment. Instruments. The hydrophilic layer is
(A) treatment with at least one plasma selected from the group consisting of hydrogen plasma, oxygen plasma and nitrogen plasma;
(B) The coating layer formed by plasma polymerization of at least one selected from the group consisting of hexamethyldisiloxane and hexadiene is subjected to the plasma treatment of (a) above, or (c) acrylic acid, propargyl alcohol, acetonitrile, The sample injection device according to claim 2, wherein the sample injection device is formed by plasma polymerizing at least one selected from the group consisting of aminoacetaldehyde dimethyl acetal, propylamine, allylamine, and pyridine.   The sample according to any one of claims 1 to 3, wherein the dry gel for first-dimensional electrophoresis is supported on a support, and the support is suspended on the lower surface of the injector body. Injection device.   5. The apparatus according to claim 1, wherein the two-dimensional electrophoresis apparatus is disposed on the apparatus main body, a second-dimensional electrophoresis gel supported on the apparatus main body, and the apparatus main body. A two-dimensional electrophoresis apparatus comprising a sample injection instrument, wherein a gap as a junction is formed between the first-dimensional electrophoresis gel and the second-dimensional electrophoresis gel, The sample injection device according to any one of claims 1 to 4, wherein the injector main body is provided with a gel liquefied material filling hole for filling a gel in a junction portion of the two-dimensional electrophoresis apparatus.   The sample injection device according to any one of claims 1 to 5, further comprising a pair of electrodes for applying a voltage to both ends of the one-dimensional electrophoresis gel. 6. The sample injection device according to claim 5, wherein only the inner wall of the sample injection port and the surface in contact with the junction filling gel are hydrophilized.   The sample injection device according to any one of claims 1 to 7, wherein the dry gel for one-dimensional electrophoresis is a pH gradient fixed gel for isoelectric focusing.   A device main body, a gel for second-dimensional electrophoresis supported on the device main body, and the sample injection instrument according to any one of claims 1 to 8 disposed on the device main body, A device for two-dimensional electrophoresis in which a gap as a junction portion is formed between the first-dimensional electrophoresis gel and the second-dimensional electrophoresis gel.   The apparatus main body includes a support base that supports the second-dimensional electrophoresis gel and a frame body that surrounds the support base, and the sample injection device is placed on the frame body. Item 10. The apparatus according to Item 9.   A buffer solution tank is provided between one end of the support base in one direction and the frame body, and the first-dimensional electrophoresis gel is positioned obliquely above one buffer solution tank. 10. The apparatus according to 10.   A step of injecting a sample into the sample injection port of the sample injection device of the apparatus for two-dimensional electrophoresis according to any one of claims 9 to 11, a step of performing a first-dimensional electrophoresis, A two-dimensional electrophoresis method including a step of filling a gel in a junction and a step of performing a second-dimensional electrophoresis.

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