JP5278060B2 - Transfer device that does not require filter paper, its production kit, and transfer method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transfer device requiring no filter paper capable of successfully performing transfer because the occurrence of air bubbles is suppressed. <P>SOLUTION: The transfer device 100 requiring no filter paper houses a separation medium 170 containing a buffer solution and a transfer film 180 and transfers an object to be transferred 171 in the separation medium 170 to the surface of the transfer film 180 by passing an electric current through the separation medium 170 through the buffer solution. The buffer solution contains an aromatic compound containing at least either one of a hydroxyl group and a carbonyl group. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention generally relates to a transfer device that does not require filter paper, and more specifically, transfers a material to be transferred in the separation medium onto the transfer film by passing an electric current through the buffer to the separation medium. The present invention relates to a transfer device that does not require filter paper.

  In order to analyze or examine biomolecules such as proteins and nucleic acids, a technique called blotting such as Western blotting may be used. In order to perform blotting, first, biomolecules are separated by electrophoresis in a separation medium such as a gel, and the separated biomolecules are transferred from the separation medium onto a transfer film. Thereby, since the biomolecule to be analyzed can be arranged on the transfer film in a separated state, further analysis can be performed successfully.

  At this time, a transfer device is used for transferring biomolecules from the separation medium to the transfer membrane. As a transfer method performed by the transfer device, a method using a capillary phenomenon, a method of applying a voltage, and a method of vacuuming are known (see Non-Patent Document 1).

  Among the methods described above, the method of applying a voltage is known to have good transfer efficiency. There are two types of transfer apparatuses that perform the method of applying a voltage, a tank type and a semi-dry type (see Non-Patent Document 2). In a tank type transfer device, a separation medium and a transfer film are held in a tank filled with a buffer solution, and a voltage is applied to the separation medium and the transfer film using an electrode provided in the tank. In the semi-dry type transfer apparatus, a filter paper immersed in a buffer solution is used, and an electrode, a filter paper, a separation medium, a transfer film, a filter paper, and an electrode are stacked in this order, and a voltage is applied between both electrodes.

JP 2008-298644 (released December 11, 2008)

Sambrook et al., Molecular Cloning, A laboratory manual, 3rd ed., Cold Spring Harbor Laboratory (2001), 6.33-6.38 Michie Oto “Q & A about electrophoresis” pages 161-163, Yodosha, 2005

  In any of the transfer methods described above, a filter paper is sandwiched between the electrode and the separation medium or transfer film (FIGS. 6-1, 6-2, and 6-3 in Non-Patent Document 1, FIG. 1 and FIG. 1 in Non-Patent Document 2). (See 2nd grade). Also in the tank type transfer device, the separation medium and the transfer film are held in the tank with the filter paper sandwiched between them (see FIG. 1 of Non-Patent Document 2). This filter paper is traditionally used and is always described in general protocol collections. For this reason, there has been no report on a transfer device that does not require filter paper.

  However, in the process of studying an apparatus (see Patent Document 1) for carrying out both electrophoresis and transfer that the present inventors previously invented, the present inventors considered that filter paper is unnecessary based on a unique viewpoint. The inventors have found that the transfer device is useful.

  That is, the apparatus described in Patent Document 1 performs both electrophoresis and transfer at the same position without transporting the separation medium. It has been found that when the separation medium is sandwiched between filter papers, the electrophoresis is inhibited by the filter paper. Based on this knowledge, the present inventors have found that it is preferable to incorporate a transfer device that does not require filter paper in the case of the device described in Patent Document 1, and further studies have been made, and a transfer device that eliminates the need for filter paper. For example, the present invention is not limited to the case of being incorporated in the apparatus described in Patent Document 1, and the process of sandwiching the filter paper is not necessary when configuring the apparatus, and the cost of the filter paper can be saved, which is advantageous over the conventional transfer apparatus. I came up with that.

  The present invention has been made in view of the above problems, and has as its main object to provide a transfer device that has not been studied so far and that does not require filter paper.

  The present inventors have intensively studied to solve the above-mentioned problems, and cannot simply provide a transfer device that does not require a practical filter paper by simply removing the filter paper from the transfer device according to the prior art. I found. That is, when the filter paper is removed from the transfer device according to the prior art, the transfer amount may be extremely reduced.

  The present inventors have conducted further studies, and the original finding that the decrease in the transfer amount due to the removal of the filter paper is caused by the current flow being blocked by bubbles generated by the electrolysis of water. Got.

  That is, when an electric current flows through the buffer solution of the transfer device, water electrolysis may occur. At this time, if a filter paper is present, bubbles generated by the electrolysis are taken into the filter paper. However, when the filter paper is removed, the bubbles may enter between the separation medium or the transfer film and the electrode. Since the gas is a nonconductor, the current flows avoiding the bubbles and the direction of the current is scattered. Therefore, the moving direction of the transferred material is also scattered, so that the transferred material is diffused and the transfer amount is reduced. In order to solve this problem, the present inventors have intensively studied and completed the present invention.

  That is, a transfer device that does not require filter paper according to the present invention stores a separation medium containing a buffer solution and a transfer film, and allows a current to flow through the buffer medium in the separation medium. A transfer device that transfers a transfer object onto the transfer film and does not require a filter paper, wherein the buffer solution contains an aromatic compound containing at least one of a hydroxyl group and a carbonyl group. It is a feature. Preferably, the transfer device includes an anode electrode and a cathode electrode for causing a current to flow through the separation medium, and the separation medium and the transfer film are disposed between the anode electrode and the cathode electrode. .

  According to the above configuration, the separation medium containing the buffer solution and the transfer film are stored, and a current is passed through the separation medium through the buffer solution, thereby transferring the transfer object in the separation medium onto the transfer film. In the transfer device that does not require filter paper to be transferred, the buffer solution contains an aromatic compound containing at least one of a hydroxyl group and a carbonyl group, so when a current is passed through the buffer solution, Since the aromatic compound is oxidized or reduced in preference to the electrolysis of water in the buffer solution, the electrolysis of water is suppressed. Thereby, it is possible to provide a transfer device that can perform transfer successfully and does not require filter paper.

  The aromatic compound is preferably hydroquinone or parabenzoquinone. The concentration of the aromatic compound in the buffer solution is preferably in the range of 0.01M to 0.4M. It is preferable that at least one of the anode electrode and the cathode electrode includes a conductor made of platinum, copper, zinc, gold, or iron.

  By using such an aromatic compound and an electrode, the transfer device can perform transfer more successfully.

  In the transfer device according to the present invention, at least one of the anode electrode and the cathode electrode may include a plurality of strip-like conductors parallel to each other.

  The plurality of strip-shaped conductors parallel to each other do not hinder the potential gradient formed in the direction orthogonal to the direction in which the strip-shaped conductor extends. Therefore, according to the above configuration, since at least one of the anode electrode and the cathode electrode does not exclude electrophoresis in the separation medium, the electrophoresis can be performed successfully. At this time, since the transfer device according to the present invention is a transfer device that does not require filter paper, there is an effect that the filter paper can perform the electrophoresis more successfully without hindering the electrophoresis.

  The transfer method according to the present invention is a transfer method in which a transfer paper in a separation medium is transferred onto a transfer film, and no filter paper is required, and the separation medium and the transfer film have at least one of a hydroxyl group and a carbonyl group. The method includes a step of including a buffer solution containing an aromatic compound containing either of them, and a step of passing an electric current through the buffer solution through the buffer solution.

  According to said structure, there can exist an effect equivalent to the transfer apparatus which concerns on this invention.

  The additive of the buffer solution used in the transfer device that does not require filter paper according to the present invention is characterized by containing an aromatic compound containing at least one of a hydroxyl group and a carbonyl group. The aromatic compound is preferably hydroquinone or parabenzoquinone.

  By preparing the buffer solution of the transfer device using such a buffer solution additive, the transfer device according to the present invention which does not require filter paper can be easily configured.

  The buffer additive according to the present invention contains both hydroquinone and parabenzoquinone, and the ratio of the content of parabenzoquinone to the content of hydroquinone is preferably ½ to 2 times. .

  By using a buffer solution prepared using such a buffer solution additive, it is possible to efficiently suppress the generation of bubbles in both the anode electrode and the cathode electrode in a transfer device that does not require filter paper. it can.

  A production kit according to the present invention is a production kit for producing a transfer device that does not require a filter paper, and transfers an object to be transferred in a separation medium onto a transfer film, the anode electrode and the cathode electrode, and the anode A storage part for storing the separation medium and the transfer membrane between the electrode and the cathode electrode; and an additive for a buffer solution containing an aromatic compound containing at least one of a hydroxyl group and a carbonyl group It is characterized by having.

  The preparation kit according to the present invention can be suitably used for constituting the transfer device according to the present invention.

  In the transfer apparatus according to the present invention, since the buffer solution contains at least one of hydroquinone and parabenzoquinone, it is possible to suppress the generation of bubbles caused by passing an electric current through the buffer solution. It is possible to provide a transfer device that can be performed successfully and does not require a filter paper.

1 is a perspective view showing a schematic configuration of a transfer apparatus according to an embodiment (first embodiment) of the present invention. 1 is a cross-sectional view illustrating a schematic configuration of a transfer apparatus according to an embodiment (first embodiment) of the present invention. It is a figure explaining the function of the transfer apparatus which concerns on one Embodiment (1st Embodiment) of this invention. It is sectional drawing which shows schematic structure of the transfer apparatus which concerns on one Embodiment (2nd Embodiment) of this invention. It is a perspective view which shows schematic structure of the transfer apparatus which concerns on one Embodiment (3rd Embodiment) of this invention. It is sectional drawing which shows schematic structure of the transfer apparatus which concerns on one Embodiment (3rd Embodiment) of this invention. It is a schematic diagram which shows schematic structure of the electrode of the transfer apparatus which concerns on one Embodiment (3rd Embodiment) of this invention. It is a figure which shows the transcription | transfer result using the transfer apparatus which concerns on one Embodiment of this invention, and the transfer apparatus used as a contrast | contrast. It is a figure which shows the transcription | transfer result using the transfer apparatus which concerns on one Embodiment of this invention, and the transfer apparatus used as a contrast | contrast.

[First Embodiment]
FIG. 1 is a perspective view showing a schematic configuration of a transfer apparatus 100 according to an embodiment (first embodiment) of the present invention. FIG. 2 is a cross-sectional view illustrating a schematic configuration of the transfer device 100. As shown in FIGS. 1 and 2, the transfer apparatus 100 includes an anode electrode 150, a cathode electrode 151, and a storage unit 110 (insulator plates 140 and 141, a spacer 142, and a holding member 143). A separation medium 170 and a transfer film 180 are stored between the cathode electrodes 151. The transfer device 100 includes a voltage applying unit 190 (not shown) for applying a voltage between the anode electrode 150 and the cathode electrode 151.

(Outline function)
FIG. 3 is a diagram for explaining the schematic function of the transfer apparatus 100. As shown in FIG. 3, the transfer medium 171 is included in the separation medium 170. In the present embodiment, the transfer object has a negative charge. Further, the separation medium 170 and the transfer film 180 include a buffer solution 160 containing a reductant (hydroquinone) 161 and an oxidant (parabenzoquinone) 162. As shown in FIG. 3, the reductant 161 and the oxidant 162 are also supplied to the vicinity of the anode electrode 150 and the cathode electrode 151 via the buffer solution.

When the voltage application unit 190 applies a voltage between the anode electrode 150 and the cathode electrode 151, a current flows in the separation medium 170 through the buffer solution 160. The voltage applying unit 190 applies a voltage to the cathode electrode 151 so that the potential of the anode electrode 150 becomes higher. That is, current flows in a direction from the transfer film 180 toward the separation medium 170 (electrons (e ⁻ ) move in the opposite direction).

  When a current flows in the separation medium 170, the transfer target 171 having a negative charge moves from the separation medium 170 to the transfer film 180 due to an electroosmosis phenomenon. As a result, the transfer object 171 can be transferred onto the transfer film 180. Note that when transferring a transfer object having a positive charge, the positional relationship between the transfer film 180 and the separation medium 170 may be reversed.

  At this time, on the anode electrode 150 and the cathode electrode 151, electrolysis of water accompanying the transfer of electrons from the electrode to the buffer solution 160 occurs, and gas is generated. Specifically, oxygen is generated at the anode electrode 150 (positive electrode), and hydrogen is generated at the cathode electrode 151 (negative electrode). The generated gas forms bubbles. As described above, the transfer is inhibited due to the bubbles. The inventors of the present invention have found that the transfer can be successfully performed even in a transfer apparatus that does not require a filter paper by preventing the generation of the bubbles.

  That is, in the transfer device (transfer device 100) according to the present invention, redox substances (reduced body 161 and oxidized body 162) are dissolved in a solution (buffer solution 160) in contact with the electrodes (anode electrode 150 and cathode electrode 151). In addition, during transfer, electrolysis of water is suppressed by transferring electrons between the redox substance and the electrode. That is, on the anode electrode 150 side, the reductant 161 donates electrons to the anode electrode 150 and becomes an oxidant 162. On the cathode electrode 151 side, the oxidant 162 receives electrons from the cathode electrode 151 and becomes a reductant 161. Thereby, generation | occurrence | production of a bubble (oxygen and hydrogen) can be suppressed.

  A desirable redox material is a material that dissolves in an aqueous solution and does not generate a gas along with redox, and more preferably a material that does not generate a solid along with redox. Further, the reaction on the anode electrode 150 side, that is, the reaction in which the reductant 161 donates electrons to the anode electrode 150 to become the oxidant 162 occurs at a potential lower than the electrolysis of water, and the reaction on the cathode electrode 151 side. It is desirable that the reaction, that is, the oxidant 162 receives electrons from the cathode electrode 151 and the reaction to become the reductant 161 occurs at a higher potential than the electrolysis of water.

  The present inventors have intensively studied a redox substance that satisfies the above-described conditions and suppresses the generation of bubbles during transfer. As a result, the redox substance contains at least one of a hydroxyl group and a carbonyl group. It has been found that aromatic compounds such as hydroquinone and parabenzoquinone are suitable. In other words, phenols are preferable as the reductant 161, and oxidants of phenols (the hydroxyl group of the phenol is substituted with a carbonyl group) as the oxidant 162 are preferable.

  That is, a filter paper that stores a separation medium and a transfer film containing a buffer solution, and transfers a transfer object in the separation medium onto the transfer film by passing an electric current through the buffer solution to the separation medium. If the transfer device is an unnecessary transfer device and the buffer solution contains an aromatic compound containing at least one of a hydroxyl group and a carbonyl group, a filter paper is unnecessary and the transfer is successful. It can be performed.

  Details of the configuration of the transfer apparatus 100 will be described below.

(Storage part)
The storage unit 110 includes insulators 140 and 141, a spacer 142, and a holding member 143. The storage unit 110 is a member for appropriately arranging the anode electrode 150, the cathode electrode 151, the separation medium 170, and the transfer film 180, and the shape thereof can be appropriately designed by those skilled in the art. The insulators 140 and 141 and the spacer 142 may be formed of an insulator. The members constituting the storage unit 110 are not limited to those in the present embodiment, and other holding members may be added, or some members may be omitted or integrated.

  A cathode electrode 151 is provided on the insulator 140. Further, an anode electrode 150 is provided on the insulator 141. The holding member 143 holds the insulators 140 and 141 so that the anode electrode 150 and the cathode electrode 151 face each other, and the cathode electrode 151, the separation medium 170, the transfer film 180, and the anode electrode 150 overlap in this order. Note that a spacer 142 may be interposed between the anode electrode 150 and the cathode electrode 151 in order to prevent the separation medium 170 from being deformed.

  1 and 2, the cathode electrode 151 is shown at the bottom, but the anode electrode 150 may be provided at the bottom. Further, as described above, the order of the separation medium 170 and the transfer film 180 may be changed depending on the property of the material to be transferred (positive or negative charge).

(Separation medium and transfer membrane)
The separation medium 170 is not particularly limited as long as it is a medium containing the transfer object 171, but is preferably a gel, and a gel generally used for electrophoresis can be used, for example, agarose gel, polyacrylamide A gel or the like can be suitably used.

  The transfer target 171 is not particularly limited, but a preparation from a biological material (for example, an individual organism, a body fluid, a cell line, a tissue culture, or a tissue fragment) can be used, and more preferably a polypeptide. Polynucleotides can also be used. These biomolecules can be easily given a negative charge by, for example, equilibration with SDS.

  The transfer film 180 is not particularly limited as long as it is made of a substance that can fix the transfer object 171, but a thin film is preferable. Specifically, as the transfer film 180, a nitrocellulose membrane, a PVDF membrane, a nylon membrane, or the like used for biopolymer analysis techniques such as a well-known Western blotting method can be suitably used.

  A buffer solution 160 is supplied between the cathode electrode 151 and the anode electrode 150. That is, the separation medium 170 and the transfer film 180 contain the buffer solution 160. As a result, a current is passed between the cathode electrode 151 and the anode electrode 150, and the transfer target 171 in the separation medium 170 can be electrophoresed and transferred to the transfer film 180. As a method of supplying the buffer solution 160, the separation medium 170 and the transfer film 180 may be preliminarily immersed in the buffer solution 160 so that the buffer solution may be included in the buffer medium 160. It may be immersed in the liquid 160. For example, the transfer medium 171 is separated in the separation medium 170 by performing electrophoresis such as SDS-PAGE using the buffer solution 160 containing at least one of the reductant 161 and the oxidant 162, The separation medium 170 may be used as it is. Furthermore, a buffer solution tank for supplying the buffer solution 160 may be provided as in second and third embodiments described later.

(Buffer solution)
The buffer solution 160 may be appropriately selected depending on the application as long as it is an electrolytic solution containing an oxidation-reduction substance that suppresses the generation of bubbles during transfer. For example, conductive materials such as Tris, CAPS, and carbonate can be used. It is preferable that a reagent having a strong buffering effect is contained.

  As described above, it is preferable to use an aromatic compound containing at least one of a hydroxyl group and a carbonyl group as a redox substance that suppresses the generation of bubbles during transfer, and it is further preferable to use hydroquinone or parabenzoquinone. preferable. The concentration of the aromatic compound in the buffer solution 160 is preferably 0.01 M or more, more preferably 0.02 M, 0.04 M, 0.06 M, or 0.08 M or more. It is preferably 4M or less, more preferably 0.35M, 0.3M, 0.25M, 0.2M or 0.15M or less.

  When the buffer 160 contains both the reductant 161 and the oxidant 162, the ratio of the concentration of the oxidant 162 to the concentration of the reductant 161 is preferably close to the same ratio. 2 times or more and 2 times or less, 2/3 times or more and 3/2 times or less, 3/4 times or more and 4/3 times or less, 4/5 times or more and 5/4 times or less, or 9/10 times or more and 10/9 times or less It can be. By making the ratio of the concentration of the oxidant 162 to the concentration of the reductant 161 close to the same factor, the generation of bubbles in both the anode electrode 150 and the cathode electrode 151 can be similarly suppressed, which is efficient.

  The buffer solution 160 may also contain a pH adjuster, a denaturant, a surfactant, alcohol, and the like. For example, when a polypeptide is transcribed, alcohol is preferably included to promote the binding between the polypeptide and the transfer film 180, and SDS is preferably included to denature the polypeptide. . In addition, when a polynucleotide is transferred, an alkaline salt such as NaOH can be used as a denaturing agent. Specific examples include, but are not limited to, Tris / glycine buffer solution, acetate buffer solution, sodium carbonate buffer solution, CAPS buffer solution, Tris / boric acid / EDTA buffer solution, Tris / acetic acid solution. / EDTA buffer solution, MOPS, phosphate buffer solution, Tris / Tricine buffer solution and other buffer solutions can be used.

(Electrode and voltage application means)
The anode electrode 150 and the cathode electrode 151 are only required to be formed of a conductive material, and are preferably materials that do not deteriorate even when brought into contact with the electrolyte used, and are not specifically limited to these. , Platinum, copper, zinc, gold, iron, and alloys thereof. As a shape, it can be made into flat plate shape, for example.

  For example, a commercially available power supply can be used as the voltage applying unit 190. What is necessary is just to electrically connect between the voltage application means 190 and the anode electrode 150 and the cathode electrode 151 by a general electric wire cord or the like.

[Second Embodiment]
FIG. 4 is a cross-sectional view showing a schematic configuration of a transfer apparatus 200 according to one embodiment (second embodiment) of the present invention. Hereinafter, the transfer device 200 will be described. However, when the same members as those of the transfer device 100 according to the first embodiment can be used, the same reference numerals are given and the description thereof is omitted.

  As shown in FIG. 4, the transfer device 200 includes a buffer solution tank 240, an anode electrode 150, a cathode electrode 151, a buffer solution 160, and a medium holding member 241. Unlike the transfer device 100, the transfer device 200 is placed vertically. Thus, the transfer device according to the present invention may be placed vertically or horizontally.

  The medium holding member 241 is formed of an insulator and is a member that holds the separation medium 170 and the transfer film 180. The medium holding member 241 has, for example, a mesh structure and can pass through the buffer solution 160.

  The buffer bath 240 stores the buffer solution 160. The buffer solution tank 240 is connected to the medium holding member 241, and the buffer solution 160 in the buffer solution tank 240 enters between the anode electrode 150 and the transfer film 180 and between the separation medium 170 and the cathode electrode 151. Or absorbed by the transfer film 180 and the separation medium 170.

  As described above, since the transfer device 200 according to the present embodiment includes the buffer solution tank 240 that supplies the buffer solution 160 to the region sandwiched between the anode electrode 150 and the cathode electrode 151, the buffer solution is easily supplied. be able to.

[Third Embodiment]
Next, the case where the transfer apparatus according to the present invention is applied to the electrophoresis and transfer apparatus described in Patent Document 1 will be described.

  FIG. 5 is a perspective view showing a schematic configuration of a transfer apparatus 300 according to one embodiment (third embodiment) of the present invention. FIG. 6 is a cross-sectional view showing a schematic configuration of the transfer apparatus 300, which is a cross-sectional view taken along the dotted line in FIG. Hereinafter, the transfer device 300 will be described, but in the case where the same members as those of the transfer device 100 according to the first embodiment can be used, the same numbers are given and the description is omitted.

  As shown in FIGS. 5 and 6, the transfer device 300 includes an anode electrode 350, a cathode electrode 351, insulators 140 and 141, and electrophoresis electrodes 352 and 353. The insulators 140 and 141 constitute the storage unit 310 and the buffer baths 320 and 321. Buffer tanks 320 and 321 store buffer 160. The storage unit 310 stores the separation medium 170 and the transfer film 180 between the anode electrode 350 and the cathode electrode 351.

  FIG. 7 is a schematic diagram showing a schematic structure of the cathode electrode 351 of the transfer device 300. As shown in FIG. 7, the cathode electrode 351 includes a plurality of strip-shaped conductors 354 that are parallel to each other, and the strip-shaped conductor 354 allows a current for transfer to flow. The direction in which the belt-like conductor 354 extends is orthogonal to the direction connecting the electrodes 352 and 353 for electrophoresis. The anode electrode 350 may be a flat electrode, but may include a plurality of strip-like conductors parallel to each other like the cathode electrode 351.

  The transfer device 300 includes a connection unit 355 and a voltage application unit 190 (not shown) for applying a voltage between the anode electrode 350 and the cathode electrode 351 and between the electrophoresis electrodes 352 and 353.

  The transfer device 300 can separate the transfer object 171 in the separation medium 170, and subsequently transfer the transfer object 171 from the separation medium 170 to the transfer film 180. That is, the transfer object 171 can be separated in the separation medium 170 by electrophoresis by including the transfer object 171 in the separation medium 170 and applying a voltage between the electrophoresis electrodes 352 and 353.

  The cathode electrode 351 includes a plurality of strip-shaped conductors 354 that are parallel to each other, but the extending direction of the strip-shaped conductor 354 is orthogonal to the direction of electrophoresis, and thus a potential gradient formed in the direction of electrophoresis. Does not disturb. Thereby, the said electrophoresis can be performed successfully. At this time, when the anode electrode 350 is a flat electrode, it is preferable to remove the anode electrode 350 together with the insulator 141 during electrophoresis.

  Here, since the transfer device 300 does not require filter paper, the electrophoresis can be performed more successfully.

  When electrophoresis is completed, transfer can be performed by applying a voltage between the anode electrode 350 and the cathode electrode 351.

(Additive for buffer)
The present invention also provides an additive for a buffer solution used in a transfer device that does not require filter paper. The additive of the buffer solution according to the present invention contains an aromatic compound containing at least one of a hydroxyl group and a carbonyl group, and can be used in, for example, the transfer devices 100, 200, and 300.

  The aromatic compound is preferably at least one of hydroquinone and parabenzoquinone, and more preferably both hydroquinone and parabenzoquinone. In particular, the ratio of parabenzoquinone to hydroquinone is preferably close to 1 ×, for example, ½ times to 2 times, 2/3 times to 3/2 times, 3/4 times to 4/3 times, 4/5 times or more and 5/4 times or less, or 9/10 times or more and 10/9 times or less. If the ratio of parabenzoquinone to hydroquinone is close to the same magnification, as described above, in the transfer device using the buffer solution according to the present invention, it is possible to obtain a uniform bubble generation suppressing effect in the anode electrode and the cathode electrode. Efficiency is good.

  The buffer additive according to the present invention may be a liquid or a solid (powder, granule, etc.), and is contained in a general buffer in addition to the aromatic compound. It may contain other ingredients such as Tris, glycine, acetic acid, EDTA, boric acid, DMSO and the like. When the additive of the buffer solution according to the present invention is a liquid, it can be diluted with distilled water or the like, for example, and the dilution factor is not particularly limited.

(Production kit)
The present invention also provides a production kit for producing the transfer device according to the present invention. The preparation kit can be, for example, a preparation kit for manufacturing the transfer apparatus 100 according to the first embodiment. The production kit for producing the transfer device 100 includes an anode electrode 150 and a cathode electrode 151, a storage unit 110 for storing the separation medium 170 and the transfer film 180 between the anode electrode 150 and the cathode electrode 151, a hydroxyl group and What is necessary is just to provide the additive of the buffer solution containing the aromatic compound containing at least any one of a carbonyl group. If such a preparation kit is used, the transfer apparatus 100 can be easily configured. In addition, the production kit according to the present invention is for producing a transfer device that does not require a filter paper according to the present invention, such as the transfer device 200 according to the second embodiment and the transfer device 300 according to the third embodiment. It can be used suitably.

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

[Example 1]
The following operations were performed for Example 1, Control Example 1 (positive control) and Control Example 2 (negative control), respectively.

(Preparation of gel)
Prepared separation gel solution (13% acrylamide mix (acrylamide: bisacrylamide = 29.2: 0.8), 375 mM Tris-HCl (pH 8.8), 0.05% APS, 0.1 in a container for gel preparation % TEMED) and added with a 7-well comb.

  After the gel (separation medium) is polymerized, SeeBlue (registered trademark) Pre-Stained Standard (invitrogen) and 0.25% agarose (ReadyPrep Overlay Agarose, BIO-RAD) used as a sample (transfer object) are 1: 1. Mix, add 15 μl per well and cool at 4 ° C. for about 15 minutes in the vertical state.

(Electrophoresis)
After the sample gelled, the sample was run for about 30 minutes at a constant current of 20 mA using a POWER PAC 3000 (BIO-RAD) as a power source.

  Here, in Control Examples 1 and 2, 25 mM Tris, 192 mM glycine + 0.1% SDS was used as a buffer for electrophoresis. On the other hand, in Example 1, 25 mM Tris, 192 mM glycine + 0.1% SDS + 50 mM parabenzoquinone + 50 mM hydroquinone was used as a buffer for electrophoresis.

(Transcription)
After the electrophoresis was completed, the gel was taken out from the gel preparation container and cut into 48 mm × 48 mm. A 0.45 μm pore membrane immersed in 1 × TGS of the same size (Membrane filter porafil (trademark registered) membrane filter, MACHEREY-NAGEL) is placed on both sides of the anode electrode so that the gel does not collapse, and both sides are anode electrodes And sandwiched between a cathode electrode and a glass plate and fixed with a large clip. At this time, in Control Example 1, a filter paper of the same size as the gel soaked in 1 × TGS was sandwiched between the anode electrode and the membrane and between the cathode electrode and the gel.

  As the power supply, Step 1: 1 V, 600 sec, Step 2: 2 V, 600 sec, and Step 3: 5 V, 2400 sec were set using KX-100L (Regulated DC Power Supply, TAKASAGO). Transfer was carried out while monitoring the current voltage value with KEITHLEY 2000 MULTITIMER. The transfer conditions were 1V for 600 seconds, 2V for 600 seconds, and 5V for 2400 seconds.

  A PPMA substrate (Pt sputtering electrode) on which Pt was sputtered was used as an anode electrode and a cathode electrode.

(detection)
The gel after transfer was photographed. FIG. 8 (a) shows the result of Control Example 1, FIG. 8 (b) shows the result of Control Example 2, and FIG. 8 (c) shows the result of Example 1.

  As shown in FIG. 8A, in Control Example 1, a dark band is transferred. On the other hand, as shown in FIG. 8B, in Control Example 2, the band is very thin. That is, when no filter paper is used, the transfer amount is greatly reduced in the conventional transfer device.

  Here, as shown in FIG. 8C, in Example 1, a sufficiently dark band was observed although no filter paper was used. In Example 1, the band interval is narrow because of the conditions during electrophoresis and is not related to the effect of the present invention.

[Example 2]
Next, it implemented about the case where a striped electrode (electrode provided with the some strip | belt-shaped conductors mutually parallel) was used as an electrode. The following operations were performed for Example 2, Control Example 3 (positive control) and Control Example 4 (negative control), respectively. In addition, except that the stripe electrode was used as the electrode and the voltage was set, Example 2 was the same as Example 1, Control Example 3 was Control Example 1, and Control Example 4 was the same procedure as Control Example 2. Conducted under conditions.

(Production of stripe electrode)
Using a sputtering apparatus (CFS-4E Para LL, Shibaura Mechatronics), an electrode was formed on a 48 mm × 66 mm PMMA plate (thickness 3 mm). Subsequently, the deposited electrode is processed using a CO2 laser engraving machine (Trotec 8010 Speed 100 C25, Trotec Co.), and the width of the conductor is about 300 μm, and the distance between the conductors is about 100 μm. A stripe electrode was produced by forming a plurality of strip-like conductors parallel to each other. Cutting (processing) conditions were set such that the laser power was 5.5%, the processing speed was 20.0%, and 1000 PPI.

(Transcription)
Except for setting the voltage, the same procedure as in Example 1 and Control Examples 1 and 2 was performed. The voltage settings were Step 1: 2 V, 600 sec, Step 2: 4 V, 600 sec, and Step 3: 10 V, 2400 sec.

(Result 2)
FIG. 9 (a) shows the result of Control Example 3, FIG. 9 (b) shows the result of Control Example 4, and FIG. 9 (c) shows the result of Example 2.

  As shown in FIG. 9A, in Control Example 1, a dark and distinct band is transferred. On the other hand, as shown in FIG. 9B, in the control example 2, the band is very thin. That is, when no filter paper is used, even when a stripe electrode is used, the transfer amount is greatly reduced in the conventional transfer device.

  Here, as shown in FIG. 9C, in Example 2, a dark band was observed as in Example 1, although no filter paper was used. In Example 2, the band interval is narrow because of the conditions during electrophoresis, and is not related to the effect of the present invention.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  The present invention can be used, for example, in the field of manufacturing instruments or devices for analyzing or examining biomolecules.

100, 200, 300 Transfer device 110, 310 Storage unit 140, 141 Insulator 142 Spacer 143 Holding member 150, 350 Anode electrode 151, 351 Cathode electrode 170 Separation medium 180 Transfer film 160 Buffer solution 161 Reductant 162 Oxidant 171 Transferred Object 190 Voltage application means 240, 320, 321 Buffer solution tank 241 Medium holding member 352, 353 Electrophoresis electrode 354 Strip conductor 355 Wiring member

Claims (7)

A separation medium and a transfer membrane containing a buffer solution are stored, and a transfer paper in the separation medium is transferred onto the transfer membrane by passing an electric current through the buffer solution to the separation medium. Transfer device,
The transfer apparatus, wherein the buffer solution contains at least one of hydroquinone and parabenzoquinone.   The transfer device according to claim 1, wherein the concentration of at least one of the hydroquinone and parabenzoquinone in the buffer solution is in a range of 0.01M to 0.4M. An anode electrode and a cathode electrode for flowing current to the separation medium are provided,
The transfer apparatus according to claim 1, wherein the separation medium and the transfer film are disposed between the anode electrode and the cathode electrode.   4. The transfer apparatus according to claim 3, wherein at least one of the anode electrode and the cathode electrode includes a plurality of strip-shaped conductors parallel to each other.   The transfer apparatus according to claim 3 or 4, wherein at least one of the anode electrode and the cathode electrode includes a conductor made of platinum, copper, zinc, gold, or iron. A transfer method that eliminates the need for filter paper to transfer the transfer object in the separation medium onto the transfer film,
Including a buffer containing at least one of hydroquinone and parabenzoquinone in the separation medium and the transfer membrane;
And a step of passing an electric current through the buffer solution through the separation medium. A production kit for producing a transfer device that does not require a filter paper, and transfers a transfer object in a separation medium onto a transfer film,
An anode electrode and a cathode electrode;
A storage section for storing the separation medium and the transfer membrane between the anode electrode and the cathode electrode; and a buffer solution additive containing at least one of hydroquinone and parabenzoquinone. A featured production kit.