JP5254184B2 - Sample separation adsorption device - Google Patents

Description

  The present invention relates to a sample separation / adsorption device for separating a biological sample into components and sequentially adsorbing the separated sample onto a member for adsorption.

  After the completion of the Human Genome Project, proteome research has been actively conducted. The term “proteome” is intended to mean all proteins that are translated and produced in specific cells, organs, and organs. Examples of such research include protein profiling.

  One of the most widely used methods for profiling proteins is protein electrophoresis, particularly two-dimensional electrophoresis. Because proteins have unique properties of charge and molecular weight, they combine a proteome, which is a mixture of many proteins, rather than separating individual proteins into their components based on charge alone or molecular weight alone. As a result, more proteins can be separated with high resolution.

  Although proteins are separated by electrophoresis and / or molecular weight by electrophoresis, it is difficult to specify biological properties from the separation position of proteins separated only by such physical properties. In addition, it is known that the function of a protein is controlled by being subjected to chemical modification (post-translational modification) such as phosphorylation after being synthesized. It is difficult to obtain information on such post-translational modifications by electrophoresis alone.

  The Western blotting method is a method of transferring a protein in a slab gel separated by electrophoresis to a membrane (see, for example, Patent Document 1). If a specific antibody is overlaid on the membrane onto which the protein has been transferred by Western blotting, the protein can be specified to some extent based on the antigen-antibody reaction. In addition, regarding phosphorylation, which is one of the post-translational modifications, it is possible to detect the presence or absence of phosphorylation and the difference in phosphorylation sites by overlaying an anti-phosphorylated protein antibody on the membrane on which the protein is transcribed Become.

  Thus, the combination of the electrophoresis method and the Western blotting method is a very useful method for specifying the biochemical properties of the protein (see, for example, Non-Patent Document 1).

  Conventionally, electrophoresis and Western blotting are performed manually by researchers using independent devices. For example, after performing isoelectric focusing and SDS-PAGE with an electrophoresis apparatus, the gel is removed from the apparatus and transferred to a transfer apparatus, and a transfer film is set and transferred (blotting). It is common to manually overlay the probe. Since the gel used for this operation is a very soft material and difficult to handle, the operation becomes complicated, and the operation requires skill.

  Therefore, a technology that automates these operations has been developed. For example, Patent Document 2 discloses a sample separation and adsorption device that automates a series of operations from electrophoresis to blotting.

Japanese Patent Laid-Open No. 7-63763 (published on March 10, 1995) JP 2007-292616 A (published on November 8, 2007)

Protein experiment note (bottom): From separation identification to functional analysis (Yodosha, 2005, paragraphs 38-47)

  Generally, by performing electrophoresis continuously for a long time, the electrode reaction changes the pH of the buffer solution, and the pH of the buffer solution changes to basic on the cathode side and acidic on the anode side. In particular, when the amount of the buffer solution is reduced in order to reduce the chip size, there is a problem that the pH change remarkably occurs.

  In the blotting method in which the separation molecules are transferred from the end face of the electrophoresis medium to the transfer membrane, it takes time to cause the high molecular weight sample to be electrophoresed to the end face. There is a problem that pH change affects electrophoresis and transcription.

  If the buffer solution on the anode side changes to an acidity of pH 4 or lower, the gel facing the buffer solution on the anode side is denatured, so that good blotting becomes difficult. In addition, if there is a significant pH gradient in the buffer, an electroosmotic flow is induced, which adversely affects the sample transfer pattern.

  In order to further increase the accuracy of sample electrophoretic separation and blotting, it is expected to realize a high-performance sample separation / adsorption device that takes into account the pH change of the buffer solution and eliminates the influence thereof.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a sample separation / adsorption device capable of transferring a sample with high accuracy.

  In order to solve the above problems, the sample separation / adsorption device according to the present study separates the sample in the separation medium by passing an electric current through the buffer to the separation medium, and the separated sample is removed. A sample separation / adsorption device for adsorbing a sample adsorption member from the separation medium, a first buffer solution tank in which a first electrode is arranged, a second buffer solution tank in which a second electrode is arranged, and the first A third buffer tank disposed between the buffer tank and the second buffer tank; a first opening that opens into the first buffer tank; and a second opening that opens into the third buffer tank. A sample separation unit having an opening and storing the separation medium; and a moving means for moving the sample adsorption member in a direction perpendicular to the sample separation direction. On the partition that separates the third buffer tank A through hole for energization that communicates the second buffer solution tank and the third buffer solution tank is provided at a portion facing the second opening. It arrange | positions between the said 2nd opening and the said through-hole in a buffer solution tank, It is characterized by the above-mentioned.

  In the above configuration, when a voltage is applied to the first electrode and the second electrode, the through hole provided in the partition wall that separates the second buffer solution tank and the third buffer solution tank, the first opening of the sample separation unit, and The buffer solution and the separation medium in the first and second buffer solution tanks are electrically connected through the second opening. At this time, the sample in the separation medium moves from the first opening toward the second opening, and is separated according to the mobility of each component.

  The separated sample is discharged from the second opening of the sample separation portion toward the through hole provided to face the second opening. Here, since the sample adsorption member is disposed between the second opening and the through hole, the sample discharged through the second opening is adsorbed (transferred) to the sample adsorption member. That is, sample adsorption is performed in a sample adsorption buffer tank in which sample adsorption members are arranged.

  At the time of sample adsorption, the sample adsorption member moves in a direction perpendicular to the sample separation direction by the moving means. For this reason, the sample discharged | emitted from 2nd opening is continuously adsorb | sucked to the member for sample adsorption sequentially.

  According to the above configuration, the sample separation / adsorption device according to the present invention performs electrophoresis using the first and second buffer solution tanks in which the electrodes are arranged, and uses the sample adsorption buffer solution tank in which no electrode is present. Sample adsorption. For this reason, even if the pH of the first buffer solution and the second buffer solution changes to acidic or basic by applying a voltage for a long time, the influence of the pH change is suppressed in the third buffer solution in the sample adsorption buffer tank. Is done.

  Therefore, in the sample separation / adsorption instrument according to the present invention, gel denaturation can be prevented, and sample adsorption can be performed in a pH environment optimal for sample adsorption. Therefore, sample adsorption to the sample adsorption member can be performed with high accuracy.

In addition, in the sample separation and adsorption device according to this research,
It is preferable that a pressing portion that presses the sample adsorbing member toward the second opening is provided on the opening of the through hole at the portion of the partition wall.

  According to the above configuration, the sample suction member is in close contact with the second opening by the pressing force of the pressing portion. Accordingly, the sample discharged from the second opening can be prevented from diffusing into the third buffer solution, and the sample can be efficiently adsorbed to the sample adsorbing member.

  Moreover, according to the said structure, since the pressing part provided on the opening of the through-hole presses the sample adsorption member, the distribution | circulation of the buffer solution through a through-hole is suppressed effectively. That is, the buffer solution in the second buffer solution tank affected by the electrode reaction can be prevented from flowing into the third buffer solution. As a result, the sample adsorption to the sample adsorption member can be performed with higher accuracy.

In addition, in the sample separation and adsorption device according to this research,
The pressing portion is preferably made of an elastic material having a through hole or a material that expands when water is absorbed.

  According to the said structure, the pressing part which ensured the electrical connection through the said through-hole can be comprised suitably.

In addition, in the sample separation and adsorption device according to this research,
The second buffer solution tank is configured as a detachable tank, and the through hole is preferably provided in a side portion of the second buffer solution tank.

  According to the said structure, the sample separation adsorption instrument which concerns on this invention can be comprised simply.

  Moreover, in the sample separation / adsorption device according to the present research, the through hole may be filled with a conductive medium.

  According to the above configuration, the circulation of the buffer solution through the through hole can be suppressed by the presence of the conductive medium itself without using the pressing portion. Further, the second opening of the sample separation unit and the sample adsorption member can be brought into close contact with each other without using the pressing unit.

  Since the sample separation / adsorption device according to the present invention includes the first and second buffer solution tanks in which the electrodes are arranged and the sample adsorption buffer solution tank in which the sample adsorption member is arranged, the sample adsorption is preferably performed under pH conditions. The sample adsorption to the sample adsorption member can be performed with high accuracy.

It is sectional drawing which shows the principal part structure of the sample separation adsorption instrument which concerns on one Embodiment of this invention. (A) is sectional drawing which shows a sample separation part roughly, (b) is the sectional drawing. (A) is sectional drawing which shows a 2nd buffer solution tank roughly, (b) is the sectional drawing. (A) is sectional drawing which shows a 2nd buffer solution tank roughly, (b) is the sectional drawing. It is sectional drawing which shows the arrangement | positioning relationship between a sample separation part and a 2nd buffer solution tank. It is a perspective view showing the principal part composition of the sample separation adsorption instrument concerning one embodiment of the present invention. It is a perspective view showing the principal part composition of the sample separation adsorption instrument concerning one embodiment of the present invention. It is sectional drawing which shows the principal part structure of the sample separation adsorption instrument which concerns on one Embodiment of this invention. It is sectional drawing which shows the sample separation part in Example 1 and 2. FIG. It is sectional drawing which shows the spacer in Example 1 and 2. FIG. It is a figure which shows the result of having performed sample adsorption | suction using the sample separation adsorption instrument which concerns on Example 1. FIG. It is a figure which shows the result of having performed sample adsorption | suction using the sample separation adsorption instrument which concerns on Example 2. FIG.

  An embodiment of the present invention will be described below with reference to the drawings.

(Configuration of sample separation / adsorption device 100)
First, a schematic configuration of the sample separation / adsorption device 100 according to the present embodiment will be described with reference to FIG. FIG. 1 is a side view schematically showing the configuration of the sample separation / adsorption device 100.

  In the following description, in the sample separation / adsorption instrument 100, the vertical direction shown in FIG. 1 is defined as the Z-axis direction, and the SDS-PAGE direction (separation direction) of the sample defined by the first electrode 41 and the second electrode 42 is defined. The direction is the Y-axis direction, and the direction perpendicular to both the Y-axis and the Z-axis is the X-axis direction.

  As shown in FIG. 1, the sample separation / adsorption instrument 100 includes a first buffer solution tank 1, a second buffer solution tank 2, a sample adsorption buffer solution tank (third buffer solution tank) 3, and a sample separation for storing a separation medium 33. A portion 31, a first electrode 41, and a second electrode 42 are provided. The sample separation / adsorption device 100 includes a moving mechanism (moving means) that moves the adsorbing member (sample adsorbing member) 6.

  Below, each member is demonstrated in detail.

  The first buffer solution tank 1 and the second buffer solution tank 2 are tanks filled with the first buffer solution and the second buffer solution, respectively, and the first electrode 41 and the second electrode 42 are arranged inside thereof. The It does not specifically limit as a 1st buffer solution and a 2nd buffer solution, According to a use or the objective, it can select suitably from the buffer solution of the composition generally used for electrophoresis. In the present embodiment, the first electrode 41 is connected to the negative electrode of a power source (not shown), and the second electrode 42 is connected to the positive electrode.

  The second buffer solution tank 2 is preferably configured as a tank that can be attached to and detached from the storage container 10. By attaching such a second buffer solution tank 2 to the storage container 10, a space between the second buffer solution tank 2, the storage container 10 and the sample separation unit 31 is realized as the sample adsorption buffer solution tank 3. In order to fix the movement of the attached second buffer solution tank 2 in the Z direction, the ascending prevention claw 80 provided in the storage container 10 may suppress the second buffer solution tank 2. As will be described in detail later, a hole 39 for energization is formed in the side portion 21 of the second buffer solution tank 2, and a pressing portion 38 and a hydrophilic film 37 are provided.

  The sample adsorption buffer tank 3 is a tank filled with a sample adsorption buffer. As the buffer solution for sample adsorption, it is preferable to use a buffer solution adjusted to a pH condition at which the sample is easily adsorbed.

  In FIG. 1, the first buffer solution tank 1 and the sample adsorption buffer solution tank 3 are realized using a partition formed in the storage container 10, a sample separation unit 31, and the like. It is not limited, and any tank that enables electrophoresis can be used.

  The sample separation unit 31 is disposed between the first buffer solution tank 1 and the sample adsorption buffer solution tank 3. At this time, the sample separation unit 31 may be placed, for example, on the sample separation unit place 8 shown in FIG. The placed sample separation unit 31 may be engaged with the sample adsorption buffer solution tank 3 in order to fix the movement in the Y direction, and in the receiving container 10 in order to fix the movement in the Z direction. It may be suppressed by the provided rise prevention claw 80.

  The sample separation unit 31 has openings at both ends in the Y-axis direction. Specifically, the sample separation unit 31 has a first opening 35 that opens into the first buffer solution tank 1 and a second opening 36 that opens into the sample adsorption buffer solution tank 3. The shapes of the first opening 35 and the second opening 36 may be rectangular, for example.

  Further, as shown in FIG. 4, the sample separation unit 31 is configured to store a separation medium 33 therein. The separation medium 33 stored in the sample separation unit 31 faces the first buffer solution tank 1 through the first opening 35 and faces the second buffer solution tank 2 through the second opening 36.

  FIG. 4A is a perspective view showing the sample separation unit 31, and FIG. 4B is a cross-sectional view thereof. As shown in FIG. 4B, the sample separation unit 31 can be composed of two insulating plates 34 and a spacer (not shown) installed to secure a space between them. On the first opening 35 side, it is desirable that the upper part of the separation medium 33 is exposed from the insulating plate 34 in order to bring the sample into contact with the separation medium 33. The hydrophilic film 32 is preferably attached to the second opening 36 in order to hold the separation medium 33 (see FIG. 5).

  The separation medium 33 is a medium for separating a sample by electrophoresis, and a medium generally used for electrophoresis, for example, polyacrylamide gel or agarose gel can be used.

  As used herein, “sample” is intended to mean “biological sample” or an equivalent thereof. A “biological sample” is intended to be any preparation obtained from biological material as a source (eg, an individual, body fluid, cell line, tissue culture or tissue section). Biological samples include body fluids (eg, blood, saliva, plaque, serum, plasma, urine, synovial fluid and cerebrospinal fluid) and tissue sources. A preferred biological sample is a subject sample. Preferred subject samples are skin lesions, sputum, pharyngeal mucus, nasal mucus, pus or secretions obtained from the subject. As used herein, the term “tissue sample” intends a biological sample obtained from a tissue source. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. As used herein, the term “sample” includes, in addition to the biological sample and the tissue sample, a protein sample extracted from the biological sample and the tissue sample, a genomic DNA sample, and / or A total RNA sample is also included. The “sample component” is intended to mean various factors (components) constituting the “sample”.

  The adsorbing member 6 is a member for adsorbing a sample, and is arranged between the second opening 36 and the hole 39 in the sample adsorption buffer tank 3 as shown in FIG. The suction member 6 is fixed to the moving arm 61 and is moved in the Z direction by a moving mechanism described later.

  The adsorbing member 6 is preferably made of a material that can ensure strength. For example, when the sample is a protein, a PVDF (Polyvinylidene fluoride) film or the like can be used. Note that the PVDF membrane is preferably hydrophilized in advance using methanol or the like. In addition, a conventionally used membrane such as nylon or nitrocellulose, which is easily bound to nucleic acid or protein, can be used.

  The first electrode 41 is disposed to face the first opening 35 in the first buffer solution tank 1, while the second electrode 42 is disposed to face the hole 39 in the second buffer solution tank 2. .

  Moreover, although the 1st electrode 41 and the 2nd electrode 42 should just be formed from an electroconductive raw material, in order to prevent ionization of an electrode, it is preferable to use platinum as a raw material. The shape of the second electrode 42 is preferably the same as the second opening 36 or smaller than the second opening 36, and may be, for example, linear.

(Operation of the sample separation / adsorption device 100)
Next, the flow up to sample adsorption in the sample separation / adsorption instrument 100 will be described with reference to FIG.

  First, the sample separation part 31 is installed in the sample separation part place 8 of the storage container 10, and the first buffer solution tank 1 is filled with the first buffer solution, and the sample adsorption buffer solution tank 3 is filled with the third buffer solution. After the sample buffer member 6 fixed to the moving arm is immersed in the third buffer solution, the second buffer solution tank is placed in the storage container 10 and then filled with the second buffer solution. Thereafter, the first electrode 41 and the second electrode 42 are disposed.

  Next, the sample is introduced into the separation medium 33. For example, when performing two-dimensional electrophoresis, the sample medium 50 that has been subjected to the first-dimensional isoelectric focusing in advance is used. When introducing the sample, the sample medium 50 is fixed to the support 51 made of acrylic or the like. The support 51 is moved by the moving arm 52, and the sample medium 50 is placed on the upper part of the separation medium 33 in the sample separation unit 31 on the first opening 35 side. In addition, when the first-dimensional electrophoresis such as isoelectric focusing is not performed, the sample may be introduced into the well formed in the separation medium 33.

  After the sample is introduced, a voltage is applied between the first electrode 41 and the second electrode 42. The 1st electrode 41 and the 2nd electrode 42 are electrically connected via each member arrange | positioned between a buffer solution and electrodes. As a result, the sample in the separation medium 33 is electrophoresed in the Y direction, separated based on the difference in mobility generated between the sample components, and discharged from the second opening 36.

  Further, after the start of electrophoresis, the moving arm 61 starts to pull up the adsorption member 6. For this reason, the sample discharged from the second opening is continuously adsorbed to the adsorbing member 6.

  Since the sample adsorption to the adsorption member 6 is performed in the sample adsorption buffer solution tank 3 where no electrode is present, it can be satisfactorily performed without being affected by the electrode reaction.

  After the sample adsorption is completed, the adsorption member 6 is collected and subjected to staining, immune reaction, or the like. Thereafter, the separation transfer pattern of the sample is detected by a fluorescence detector or the like.

(Second buffer tank)
Next, the configuration of the second buffer solution tank 2 will be described in detail.

  FIG. 3A is a perspective view showing the second buffer solution tank 2, and FIG. 3B is a cross-sectional view thereof. 4A is a perspective view showing the second buffer solution tank 2 provided with the pressing portion 38 and the hydrophilic film 37, and FIG. 4B is a cross-sectional view thereof.

  As shown in FIGS. 3A and 3B, the side (part of the partition wall) 21 of the second buffer solution tank 2 has a hole (through hole) 39 for energizing the inside and outside of the tank in the Y direction. Is formed. The shape of the opening of the hole 39 can be formed in a rectangular shape, for example.

  When the second buffer solution tank 2 is filled with the second buffer solution, the inside of the hole 39 is filled with the second buffer solution. When the hole 39 is filled with the buffer solution, it is possible to stably apply a voltage between the first electrode 41 and the second electrode 42.

  Also, as shown in FIGS. 4A and 4B, it is preferable that a pressing portion 38 is installed on the side portion 21 of the second buffer solution tank 2 on the opening of the hole 39. Further, it is more preferable that the hydrophilic film 37 is bonded onto the pressing portion 38. The pressing portion 38 preferably has a configuration that does not hinder the electrical connection between the first electrode 41 and the second electrode 42 and is capable of pressing the suction member 6 against the second opening 36.

  For example, the pressing portion 38 can be made of a material that expands when water is absorbed, an elastic material having a through hole, or the like. Specific examples of the pressing unit 38 include filter papers having different thicknesses and compression degrees, polyvinyl alcohol, vinylon, polyvinyl acetate, and polyurethane.

  Moreover, as the hydrophilic film | membrane 37, a hydrophilic PVDF film | membrane etc. can be used, for example.

  FIG. 5 is a diagram showing a preferable arrangement relationship between the second buffer tank 2 and the sample separation unit 31.

  As shown in FIG. 5, the second buffer solution tank 2 and the sample separation unit 31 are arranged so as to sandwich the adsorption member 6. At this time, the hole 39 of the second buffer solution tank 2 faces the second opening 36 of the sample separation unit 31.

  The second buffer solution tank 2 is configured such that a pressing portion 38 provided in the second buffer solution tank 2 presses the adsorbing member 6 toward the second opening 36. For example, the second buffer solution tank 2 may be fixed to the storage container 10 so that the pressing portion 38 presses the adsorbing member 6 using the elasticity and expandability of the material. Due to the pressing force, the adsorbing member 6 is in close contact with the second opening 36 of the sample separation unit 31 and also in close contact with the side portion 21 (more specifically, the hydrophilic film 37) of the second buffer solution tank 2. To do. The hydrophilic film 37 plays a role of further improving the adhesion, and also plays a role of improving slipping when the adsorption member 6 moves in the Z-axis direction.

  Here, when the second opening 36 and the adsorbing member 6 are in close contact with each other, it is possible to prevent the sample discharged from the second opening 36 from diffusing into the third buffer solution. As a result, the sample can be efficiently adsorbed to the adsorbing member 6.

  Further, when the side portion 21 of the second buffer solution tank 2 and the adsorbing member 6 are in close contact with each other, the flow of the buffer solution through the hole 39 is effectively suppressed. That is, the second buffer solution affected by the electrode reaction is suppressed from flowing into the buffer solution in the sample adsorption buffer solution tank 3. As a result, the sample can be adsorbed to the adsorbing member 6 with higher accuracy.

  In the present invention, the hole 39 can be filled with a conductive medium such as a gel instead of the buffer solution. However, when the hole 39 is filled with a conductive medium such as gel, as soon as the electrical connection cannot be obtained due to the influence of the voltage application for a long time, the voltage value rapidly rises and the adsorption stops. Therefore, in order to stably apply a voltage for a long time, it is preferable to fill the hole 39 with a buffer solution.

  Further, the present invention is not limited to the configuration in which the pressing portion 38 is provided on the side portion 21 of the second buffer solution tank 2. For example, the side portion 21 provided with the pressing portion 38 is not limited to the side wall of the second buffer solution tank 2 and may be a partition wall that separates the second buffer solution tank 2 and the sample adsorption buffer solution tank 3.

  Even if the pressing portion 38 is not provided, the flow of the buffer solution between the second buffer solution tank 2 and the sample adsorption buffer solution tank 3 is such that the sample adsorbing member 6 and the side portion 21 are in close contact with each other. The effect of the present invention can be obtained by being limited to the ones that pass through.

  Moreover, the sample separation / adsorption instrument 100 according to the present embodiment may be provided in a state in which the above-described members are attached in advance, or may be provided in a configuration in which the user attaches each member later.

(Movement mechanism of adsorption member 6)
Next, an example of a moving mechanism that moves the adsorption member 6 will be described.

  6 and 7 are perspective views showing a moving mechanism provided in the sample separation / adsorption instrument 100. FIG. Further, in FIG. 7, the moving arm 52 and the support body 51 are omitted in order to simplify the drawing.

  The entire sample separation / adsorption device 100 including the driving means 7 is fixed to the base 9. The adsorbing member 6 is fixed to the moving arm 61, and the moving arm 61 is driven by the driving means 7. Specifically, the moving arm 61 is connected to the first driving means 71, and the first driving means 71 is connected to the second driving means 72 fixed on the base 9. The moving arm 61 is driven in the Y direction and the Z direction by the first driving means 71 and the second driving means 72. Thereby, the movement of the adsorption member 6 can be controlled.

  FIG. 8 is a cross-sectional view showing the sample separation / adsorption device 100 in order to explain the control between the driving means 7 and the electrodes (the first electrode 41 and the second electrode 42).

  As shown in FIG. 8, the first electrode 41 and the second electrode 42 are connected to the negative electrode and the positive electrode of the power supply 111 via the wiring 112, respectively. An ammeter 113 is disposed between the power source 111 and the second electrode 42. The data processing device 114 controls the power supply 111 so that a constant current flows between the first electrode 41 and the second electrode 42.

  Further, as shown in FIG. 8, the driving means 7 (first driving means and second driving means) is controlled by a driving means control unit 70. When it is time to pull up the suction member 6, the drive means controller 70 controls the drive means 7 to start driving. Further, the data processing device 114 may control the driving means control unit 70 based on the current value measured by the ammeter 113.

  Although not shown, the moving arm 52 can also be driven using the same means as the moving mechanism described above, whereby the introduction of the sample can be controlled.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to a following example.

Example 1
In Example 1, the second separation electrophoresis and sample adsorption of two-dimensional electrophoresis were performed using the sample separation / adsorption instrument 100 according to the present embodiment.

[First-dimensional electrophoresis (sample medium 50)]
As the sample medium 50, a gel for immobilizing pH gradient isoelectric focusing by immobiline was cut into 1 mm × 60 mm and used. Sample introduction and gel swelling were performed, and electrophoresis was performed at 6000 V for 1 hour.

  As a sample, a mouse liver sample stained with a fluorescent marker was used.

[Sample separation unit 31]
As the two insulating plates 34, quartz glass having a thickness of 60 mm × 30 mm × 2 mm and a thickness of 60 mm × 28 mm × 2 mm, both having 0.5 mm steps at both ends was used. A hydrophilic PVDF film was attached to the outside of the two quartz glasses with Kapton tape. At this time, care was taken so that there was no gap between the hydrophilic PVDF film and the glass surface, and the Kapton tape was made not to inhibit the second opening 36. Both ends of the quartz glass protrude 0.5 mm from the center, and the thickness of the separation medium is defined from the protrusion.

  A sample separation unit 31 prepared as described above and filled with polyacrylamide gel was placed in the sample separation unit place 8 and fixed with a rise prevention claw 80.

[First buffer tank 1]
The first buffer solution tank 1 of 70 mm × 70 mm × depth 15 mm (depth 8 mm in the sample separation part storage 8) was provided in the container 10 and filled with the first buffer solution. A commercially available MOPS / SDS buffer was used as the first buffer solution. A platinum wire was used as the first electrode 41 disposed in the first buffer tank 1. The first electrode 41 was connected to the negative electrode of the power source 111.

[Second buffer tank 2]
The second buffer solution tank 2 of 70 mm × 70 mm × depth 15 mm was used, and a pressing part 38 was installed outside the side part 21 (1 mm thickness in the Z direction) of the tank. A hydrophilic film 37 was attached to the surface of the pressing portion 38. The second buffer solution tank 2 was installed by being fitted into the storage container 10, and fixed by the rise prevention claw 80. The second buffer solution 2 was filled in the second buffer solution. A commercially available MOPS buffer was used as the second buffer solution. A platinum wire was used as the second electrode 42 disposed in the second buffer solution tank 2. At this time, the second electrode 42 was connected to the positive electrode of the power source 111.

[Sample adsorption buffer tank 3]
The space formed between the second buffer solution tank 2, the storage container 10, and the sample separation unit 31 by installing the second buffer solution tank 2 in the storage container 10 was used as the sample adsorption buffer solution tank 3. The sample adsorption buffer tank 3 was filled with a third buffer solution having a pH condition that facilitates adsorption of the observation target sample. As the third buffer, a commercially available MOPS buffer was used as in the second buffer.

[Adsorption member 6]
As the adsorbing member 6, a hydrophobic PVDF hydrophilized with methanol was used. The upper end side of the adsorption member 6 was fixed to the moving arm 61, and the lower end side was immersed in the third buffer solution in the sample adsorption buffer solution tank 3.

[Introduction of the sample medium 50 into the separation medium 33]
The sample medium 50 containing the first-dimensional electrophoresis sample is brought into close contact with the portion where the separation medium 33 is exposed in the first opening 35 of the sample separation section 31 by the moving arm 52.

[Second-dimensional electrophoresis]
With the sample medium 50 and the separation medium 33 in close contact, a constant current was passed between the first electrode 41 and the second electrode 42 for 25 minutes so that the value of the ammeter 113 was 25 mA.

[Sample adsorption]
A constant current was passed between the first electrode 41 and the second electrode 42 for 60 minutes so that the value of the ammeter 113 was 40 mA. At this time, the first driving means 71 lifted the suction member 6 upward. The pulling speed was gradually reduced from 32 μm / sec to 2 μm / sec by the drive means controller 70.

〔result〕
After adsorbing the sample, the adsorbing member 6 was collected and the sample was detected. The detection result is shown in FIG. As shown in FIG. 11, according to Example 1, it was possible to obtain a sample adsorption pattern with high resolution while continuously performing sample separation and sample adsorption with the same instrument.

(Example 2)
In Example 2, SDS-PAGE and sample adsorption were performed using the sample separation / adsorption device 100 according to the present embodiment. In the second embodiment, the description of the same as the first embodiment is omitted, and only the differences will be described below.

[Sample separation unit 31]
The sample separation unit 31 was produced in the same manner as in Example 1. The sample separation unit 31 was filled with polyacrylamide gel as the separation medium 33. Before the polyacrylamide gel was polymerized, a comb was inserted into the upper portion of the first opening 35 to prepare a well for introducing a one-dimensional electrophoresis sample. 10 μL of a molecular weight visible marker sample was poured into the prepared well, and then the well was covered with an agarose gel. A hydrophilic film 37 was wound around the second opening 36.

  As a sample, a visible protein marker was used.

[SDS-PAGE]
A constant current was passed between the first electrode 41 and the second electrode 42 for 25 minutes so that the value of the ammeter 113 was 25 mA.

[Sample adsorption]
A constant current was passed between the first electrode 41 and the second electrode 42 for 60 minutes so that the value of the ammeter 113 was 40 mA. At this time, the first driving means 71 lifted the suction member 6 upward. The pulling speed was gradually reduced from 32 μm / sec to 2 μm / sec by the drive means controller 70.

〔result〕
After adsorbing the sample, the adsorbing member 6 was collected and the sample was detected. The detection result is shown in FIG. As shown in FIG. 12, according to Example 2, a sample adsorption pattern with high resolution could be obtained while performing sample separation and sample adsorption continuously with the same instrument.

  The present invention can further develop proteome research that is being actively conducted. Moreover, the market can be activated by separately producing and selling the sample separation / adsorption device according to the present invention and various members used in the device.

DESCRIPTION OF SYMBOLS 1 1st buffer tank 2 2nd buffer tank 3 Sample adsorption buffer tank (3rd buffer tank)
6 Adsorption member (sample adsorption member)
7 Driving means 21 Side portion 31 Sample separation portion 33 Separation medium 35 First opening 36 Second opening 38 Pushing portion 41 First electrode 42 Second electrode 61 Moving arm 100 Sample separation adsorption device

Claims (5)

A sample separation / adsorption instrument for separating a sample in the separation medium by passing an electric current through the buffer to the separation medium and adsorbing the separated sample from the separation medium to a sample adsorption member,
A first buffer tank in which the first electrode is disposed;
A second buffer solution tank in which the second electrode is disposed;
A third buffer tank disposed between the first buffer tank and the second buffer tank;
A first separation opening in the first buffer solution tank and a second opening in the third buffer solution tank, and a sample separation unit for storing the separation medium,
The partition that separates the second buffer tank and the third buffer tank is filled with the buffer filled in the second buffer tank at a position facing the second opening. Through holes for
The sample separating and adsorbing instrument, wherein the sample adsorbing member is disposed between the second opening and the through hole in the third buffer tank. A pressing portion that presses the sample adsorbing member against the second opening;
The sample separation adsorption device according to claim 1, wherein the pressing portion is provided between the through hole and the sample separation portion.   The sample separating and adsorbing device according to claim 2, wherein the pressing portion is formed of an elastic material having a through hole or a material that expands when water is absorbed. The second buffer solution tank is configured as a detachable tank,
4. The sample separation / adsorption device according to claim 1, wherein the through hole is provided in a side portion of the second buffer solution tank. 5. The sample separation / adsorption device according to any one of claims 1 to 4 , further comprising moving means for moving the sample adsorption member in a direction perpendicular to a sample separation direction.