JP2010216862A - Transfer device requiring no filter paper - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transfer device requiring no filter paper, capable of simply obtaining good transfer results, even if an air bubble absorbing means such as the filter paper is not used. <P>SOLUTION: The transfer device 100 is constituted so as to transfer a sample to the transfer film 103 arranged in contact with a sample-containing gel 102 by applying voltage to the gel 102 through an electrolyte. The anode electrode 104 of a pair of electrodes consisting of the anode electrode 104 and a cathode electrode 101 contains iron. Since iron contained in the anode electrode 104 acts as a sacrifice oxidizing agent when transferred, the occurrence of air bubbles caused by the electrolysis of the electrolyte is suppressed. <P>COPYRIGHT: (C)2010,JPO&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. Is not limited to the case of being incorporated into the apparatus described in Patent Document 1, but is advantageous over the conventional transfer apparatus in that the process of sandwiching the filter paper is not required when the apparatus is configured, and the cost of the filter paper can be saved. 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.

  In order to solve the above-described problems, a transfer device according to the present invention stores a separation medium and a transfer film, and causes the material to be transferred in the separation medium to pass through the separation medium by passing a current through a buffer solution. A transfer device that does not require a filter paper to be transferred onto a transfer film, and includes an anode electrode and a cathode electrode that are a pair of electrodes for flowing the current, and at least a part of the anode electrode is made of iron. It is characterized by containing.

  According to the above configuration, at least a part of the anode electrode is made of iron. Iron is a substance that does not dissolve in the electrolyte and does not generate gas with the oxidation reaction, and does not form a passive state with the oxidation reaction. Furthermore, iron is a substance having a standard potential lower than 0 and a potential at which an anodic reaction (oxidation reaction) occurs is lower than an anode potential at the time of electrolysis of water. Since it is a substance, when a voltage is applied, iron constituting the anode electrode releases electrons as a sacrificial oxidant, thereby suppressing electrolysis of the buffer solution (water) on the anode electrode side. This suppresses the generation of bubbles from the anode electrode during transfer.

  In the transfer device according to the present invention, the transfer is performed with the separation medium and the transfer film stored between the cathode electrode and the anode electrode. Here, the separation medium is disposed on the cathode electrode side, and the transfer film is disposed on the anode electrode side. Therefore, since the generation of bubbles is suppressed on the anode electrode on the side where the transfer film exists, bubbles generated in the vicinity of the transfer film are suppressed.

  Therefore, according to the transfer apparatus according to the present invention, a good transfer result can be easily obtained without obstructing the transfer by the bubbles without using a means for absorbing bubbles such as filter paper.

  In the transfer device according to the present invention, the surface of the anode electrode that contacts the transfer film is preferably made of iron.

  According to the said structure, it can suppress effectively that a bubble generate | occur | produces from an anode electrode at the time of transcription | transfer.

  In the transfer apparatus according to the present invention, the anode electrode is preferably made of iron.

  According to the said structure, an anode electrode can be comprised more simply.

  In the transfer device according to the present invention, at least one of the anode electrode and the cathode electrode may be formed in a plate shape.

  According to the said structure, it can transfer suitably by pinching | interposing a separation medium and a transfer film between the said electrodes.

  In the transfer device according to the present invention, it is preferable that at least one of the anode electrode and the cathode electrode is formed in a plurality of parallel strips.

  According to the above configuration, when the transfer device according to the present invention is applied to an apparatus that performs both electrophoresis and transfer at the same position without transporting the separation medium, electrophoresis is not inhibited by the filter paper. And transfer can be suitably performed.

  Further, the transfer method according to the present invention stores the separation medium and the transfer film, and transfers the material to be transferred in the separation medium onto the transfer film by flowing an electric current through the buffer to the separation medium. A transfer method that eliminates the need for filter paper is characterized in that at least a part of the anode electrode, which is one of the pair of electrodes through which the current flows, is made of iron.

  According to the above method, the same effect as that of the transfer device according to the present invention can be obtained.

  The transfer device according to the present invention includes an anode electrode and a cathode electrode, which are a pair of electrodes for applying a voltage. Since the anode electrode contains iron, the generation of bubbles in the anode electrode is suppressed. A good transfer result can be easily obtained without using means for absorbing bubbles such as filter paper.

1 is a schematic diagram illustrating a transfer apparatus according to an embodiment of the present invention. 1 is a schematic diagram illustrating a transfer apparatus according to an embodiment of the present invention. 1 is a schematic diagram illustrating a transfer device according to Embodiment 1. FIG. 6 is a schematic diagram showing a transfer apparatus according to Comparative Example 1. FIG. 10 is a schematic diagram showing a transfer apparatus according to Comparative Example 2. FIG. FIG. 6 is a diagram illustrating a transfer result by the transfer device according to the first embodiment. It is a figure which shows the transfer result by the transfer apparatus which concerns on the comparative example 1. FIG. It is a figure which shows the transfer result by the transfer apparatus which concerns on the comparative example 2. FIG. FIG. 6 is a schematic diagram illustrating a transfer device according to a second embodiment. 10 is a schematic diagram showing a transfer apparatus according to Comparative Example 3. FIG. 10 is a schematic diagram showing a transfer device according to Comparative Example 4. FIG. FIG. 10 is a diagram illustrating a transfer result by a transfer device according to a second embodiment. It is a figure which shows the transfer result by the transfer apparatus which concerns on the comparative example 3. It is a figure which shows the transfer result by the transfer apparatus which concerns on the comparative example 4.

  Hereinafter, embodiments of the present invention will be described in detail.

  The present invention is a transfer apparatus for applying a voltage to a separation medium containing a sample via an electrolytic solution and transferring the sample to a transfer film disposed in contact with the separation medium. Among these electrodes, the anode electrode provides a transfer device containing iron.

  In this specification, the term “transfer” is intended to move a sample contained in a specific medium from the medium to another medium.

  FIG. 1 is a schematic diagram showing an outline of a transfer apparatus 100 according to an embodiment of the present invention. As shown in FIG. 1, the transfer device 100 according to the present invention includes a cathode electrode 101 and an anode electrode 104, and a gel (between the cathode electrode 101 and the anode electrode 104 at the time of transfer by the transfer device 100. Separation medium) 102 and transfer film 103 are arranged to overlap each other.

  The gel 102 is not particularly limited as long as it is a medium containing a sample. For example, an agarose gel, a polyacrylamide gel, or the like, which is generally used for electrophoresis and can contain a sample, is preferably used. Can do.

  Although it does not restrict | limit especially as a sample, The preparation etc. from biological material (For example, a living individual, a bodily fluid, a cell line, a tissue culture, or a tissue fragment) etc. can be used, More preferably, polypeptide or polynucleotide Can be used.

  The transfer film 103 is not particularly limited as long as it is made of a substance that can fix the sample, and a thin film is preferable. Specifically, as the transfer film 103, 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. The transfer film 103 is held in contact with the gel 102.

  A buffer solution is supplied between the cathode electrode 101 and the anode electrode 104. As a result, a current can be passed between the cathode electrode 101 and the anode electrode 104, and the sample in the gel 102 can be electrophoresed and transferred to the transfer film 103. As a method for supplying the buffer solution, the whole or a part of the transfer device 100 may be immersed in the buffer solution, or the gel 102 and the transfer film 103 may be preliminarily immersed in the buffer solution so that the buffer solution is contained therein. You may not.

  The buffer solution may be appropriately selected depending on the application. For example, it is preferable that a reagent having a strong buffering effect such as Tris, CAPS, and carbonate is included. The electrolytic solution may also contain a pH adjuster, a modifier, a surfactant, alcohol, and the like. For example, when a polypeptide is transcribed, an alcohol is preferably included to promote the binding between the polypeptide and a transfer medium, and SDS is preferably included to denature the polypeptide. . In addition, when a polynucleotide is transferred, an alkali 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.

  The cathode electrode 101 only needs to be formed of a conductive material, and is preferably a material that does not deteriorate even when it is brought into contact with the electrolytic solution to be used. , Zinc or the like.

  On the other hand, the anode electrode 104 is made of iron. The whole anode electrode 104 may be iron, but is not limited thereto, and may be formed so as to include at least iron so that the iron comes into contact with the buffer solution. For example, only the surface portion of the anode electrode 104 may be iron, or iron may be formed in a stripe shape on an insulator. For example, the material of the anode electrode 104 may be an alloy containing iron.

  Iron is a substance that does not dissolve in the buffer and does not generate a gas during the oxidation reaction, does not form a non-moving body with the oxidation reaction, and has a potential lower than the anode potential during electrolysis of water. In this case, the anode reaction takes place.

  For example, the anode electrode 104 and the cathode electrode may be formed in a plate shape.

  When transfer is performed using the transfer device 100, the gel 102 containing the sample and the transfer film 103 are disposed so as to overlap each other between the cathode electrode 101 and the anode electrode 104. At this time, it is preferable that the gel 102 is disposed on the cathode electrode 101 side and the transfer film 103 is disposed on the anode electrode 104 side. Next, a buffer solution is supplied between the cathode electrode 101 and the anode electrode 104, and a voltage is applied between the electrodes. What is necessary is just to set the voltage to apply suitably according to the sample which should be transferred. When a voltage is applied between the electrodes, the sample in the gel 102 is electrophoresed, moves to the transfer film 103, and is transferred.

  At the time of transfer, the anode electrode 104 functions as a sacrificial oxidant, and suppresses electrolysis of the buffer solution at the anode electrode 104. Thereby, in the anode electrode 104, the generation of oxygen due to the electrolysis of the buffer solution can be suppressed. As a result, it is possible to suppress the bubbles existing between the anode electrode 104 and the transfer film from acting as an insulator and disturbing the potential distribution.

  In the transfer device according to the prior art, filter paper is sandwiched between the cathode electrode and the gel and between the anode electrode and the transfer film, and bubbles generated in the electrode are absorbed by the filter paper.

  On the other hand, in the transfer apparatus 100 according to the present invention, it is possible to sufficiently improve transfer efficiency without using means for absorbing bubbles such as filter paper. Therefore, according to the transfer device 100, the trouble of arranging the filter paper in the transfer device is omitted, and a good transfer result can be easily obtained.

  In addition, when assembling the arrangement for transfer by overlapping gels or transfer films between the electrodes, the amount of bubbles contained between the electrodes by the above assembly can be reduced as compared with the case where the filter paper is sandwiched.

  Since the anode electrode 104 is in an oxidized state after the transfer, only the anode electrode 104 in the transfer device 100 may be replaced at the next transfer.

(Electrophoresis and transfer device)
In one embodiment, the transfer device 100 according to the present invention can be used in an electrophoresis and transfer device.

  FIG. 2 is a diagram illustrating a schematic configuration of the transfer device 120 according to the present embodiment. The transfer device 120 performs both electrophoresis and transfer at the same position without conveying the separation medium.

  As shown in FIG. 2, the transfer device 120 includes the configuration of the transfer device 100 according to the present invention, that is, a cathode electrode 101 and an anode electrode 104. Further, the transfer device 120 includes a buffer solution tank 108 provided with an electrode 106 and a buffer solution tank 109 provided with an electrode 107.

  Here, the electrodes 106 and 107 constitute first voltage applying means, and the cathode electrode 101 and the anode electrode 104 constitute second voltage applying means. As shown in FIG. 2, the direction (first direction) in which the voltage of the first voltage application unit is applied is orthogonal to the direction (second direction) in which the voltage of the second voltage application unit is applied.

  Further, the cathode electrode 101 and the anode electrode 104 are divided into regions of a separation part 111 and a connection part 112. In the separation unit 111, the cathode electrode 101 and the anode electrode 104 hold the gel 102 and the transfer film 103. Moreover, in the connection part 112, the connection instrument 110 switches connection and disconnection to the power supply of the cathode electrode 101 and the anode electrode 104.

  In the transfer device 120, the connection of the first voltage means and the second voltage means to the respective power sources is controlled. First, the sample in the gel 102 is separated in the first direction using only the first voltage application means, The separated sample in the gel 102 can be transferred to the transfer film 103 using only the second voltage applying means.

  In the transfer device 120, the cathode electrode 101 and the anode electrode 104 are preferably formed in a plurality of strips that are insulated from each other and arranged in parallel along the first direction, that is, in the form of stripes. As a result, even when the first voltage applying means applies a voltage between the electrodes 106 and 107, the plurality of electrode regions are insulated from each other, and therefore can have different potentials. Since they are arranged along one direction, a potential gradient can be formed in the first direction. Thus, the electrodes 106 and 107 do not interfere with the electrophoresis in the second direction.

  Further, at the time of transfer, the plurality of electrode regions are connected by an appropriate wire connection device 110, so that the second voltage applying unit can apply a voltage between the cathode electrode 101 and the anode electrode 104.

  In the transfer device 120 according to the present embodiment, since it is not necessary to sandwich the gel 102 and the transfer film 103 with a filter paper for transfer, electrophoresis by the first voltage means is not hindered by the filter paper. Therefore, according to the transfer device 120, both electrophoresis and transfer can be suitably performed.

  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.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

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

[Example 1]
As Example 1, the transfer device 100 was manufactured using a Pt sputtering electrode (Pt sputtering electrode) based on a PMMA plate as the cathode electrode 101 and an iron plate electrode having a thickness of 0.5 mm as the anode electrode 104. The structure of Example 1 is shown in FIG.

  As Comparative Example 1, a transfer device 200 was manufactured using Pt sputtering electrodes for the cathode electrode 201 and the anode electrode 204. The structure of Comparative Example 1 is shown in FIG. In Comparative Example 1, filter paper 206 was disposed between the gel 202 and the cathode electrode 201 and between the transfer film 203 and the anode electrode 204. The filter paper 206 was the same size as the gel 202, and what was immersed in 1 × TGS was used.

  As Comparative Example 2, a transfer device 300 was manufactured using Pt sputter electrodes for the cathode electrode 301 and the anode electrode 304. The structure of Comparative Example 1 is shown in FIG. Comparative Example 2 has the same configuration as Comparative Example 1 except that no filter paper is disposed in the transfer device 300.

  The transfer described below was performed using the transfer apparatuses of Example 1, Comparative Example 1, and Comparative Example 2, respectively.

(sample)
SeeBlue (registered trademark) Pre-Stained Standard (invitrogen) was used as a sample for transcription.

(Electrophoresis: Preparation of gel before transfer)
The prepared separation gel solution (13% acrylamide mix (acrylamide: bisacrylamide = 29.2: 0.8), 375 mM Tris-HCl (welded with a welding chip for a fully automatic two-dimensional electrophoresis apparatus welded with an ultrasonic pressure welder) pH 8.8), 0.05% APS, 0.1% TEMED) was added and the well was capped with a 7-well comb.

  After the gel was polymerized, the sample and 0.25% agarose (ReadyPrep Overlay Agarose, BIO-RAD) were mixed 1: 1, and 15 μl per well was added and cooled at 4 ° C. for about 15 minutes in the vertical state. . After the sample gelled, it was migrated for about 30 minutes at a constant current of 20 mA setting using POWER PAC 3000 (BIO-RAD) as a power source.

  Moreover, 25 mM Tris, 192 mM glycine, and 0.1% SDS were used for the electrophoresis buffer composition.

(Transcription)
The gel was taken out from the welding tip for a fully automatic two-dimensional electrophoresis apparatus after the electrophoresis was completed, and cut out into 48 mm × 48 mm. Layered with 0.45 μm cellulose membrane (Membrane filter porafil (registered trademark) membrane filter, MACHEREY-NAGEL) soaked in 1 × TGS of the same size. Further, it was sandwiched between glass plates and fixed with a large clip.

  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.

(detection)
The gel after transfer was photographed. The transfer results obtained by the transfer apparatuses of Example 1, Comparative Example 1, and Comparative Example 2 are shown in FIGS.

  FIG. 5 is a diagram illustrating a transfer result according to the first embodiment. FIG. 6 is a diagram illustrating a transfer result according to Comparative Example 1. 10 is a diagram showing a transfer result according to Comparative Example 2. FIG.

  Comparing FIGS. 5 to 7, according to Example 1, it was found that transfer was performed well without using filter paper. As shown in FIG. 5, in the result of Example 1, rust caused by the iron of the anode electrode adhered to a narrow region of the outer peripheral portion, but no rust adhered to the central portion.

[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 stripe electrode was produced according to the following procedure and conditions.

(Production of stripe electrode)
Using a sputtering apparatus (CFS-4EP-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. The cutting (processing) conditions were a laser power of 5.5%, a processing speed of 20.0%, and 1000 PPI.

  As Example 2, an electrode (Pt stripe electrode) in which a plurality of platinum strip conductors parallel to each other are formed on a PMMA plate is used for the cathode electrode 401, and a plurality of parallel electrodes on the PMMA plate are used for the anode electrode 104. The transfer device 400 was manufactured using an electrode (Fe stripe electrode) on which an iron strip conductor was formed. The structure of Example 2 is shown in FIG.

  As Comparative Example 3, a transfer device 500 was manufactured using Pt stripe electrodes for the cathode electrode 501 and the anode electrode 504. The structure of Comparative Example 3 is shown in FIG. In Comparative Example 3, filter paper 506 was disposed between the gel 502 and the cathode electrode 501 and between the transfer film 503 and the anode electrode 504. The filter paper 506 was the same size as the gel 502, and what was immersed in 1xTGS was used.

  As Comparative Example 4, a transfer device 600 was manufactured using Pt sputtering electrodes for the cathode electrode 601 and the anode electrode 604. The structure of Comparative Example 4 is shown in FIG. Comparative Example 4 has the same configuration as Comparative Example 3 except that no filter paper is disposed in the transfer device 600.

  Using the transfer devices of Example 2, Comparative Example 3, and Comparative Example 4 described above, transfer was performed in the same procedures and conditions as in Example 1, Comparative Example 1, and Comparative Example 2. However, the voltage settings were Step 1: 2 V, 600 sec, Step 2: 4 V, 600 sec, and Step 3: 10 V, 2400 sec.

  FIG. 12 is a diagram illustrating a transfer result according to the second embodiment. FIG. 13 is a diagram illustrating a transfer result according to Comparative Example 3. FIG. 14 is a diagram illustrating a transfer result according to Comparative Example 4.

  When comparing FIGS. 12 to 14, the transfer amount of the sample in Example 2 is not as high as that of Comparative Example 3, but the transfer amount is particularly low in the low molecular protein (lower gel) as compared with Comparative Example 4. It was a lot.

  The present invention can be used, for example, in the field of manufacturing instruments for life science research.

DESCRIPTION OF SYMBOLS 100 Transfer apparatus 101 Cathode electrode 102 Gel 103 Transfer film 104 Anode electrode

Claims (6)

A transfer device that stores a separation medium and a transfer film, and 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. ,
An anode electrode and a cathode electrode, which are a pair of electrodes for flowing the current,
At least a part of the anode electrode contains iron.   2. The transfer apparatus according to claim 1, wherein a surface of the anode electrode that contacts the transfer film is made of iron.   The transfer device according to claim 1, wherein the anode electrode is made of iron.   4. The transfer device according to claim 1, wherein at least one of the anode electrode and the cathode electrode is formed in a plate shape. 5.   4. The transfer device according to claim 1, wherein at least one of the anode electrode and the cathode electrode is formed in a plurality of parallel strips. 5. A transfer method that eliminates the need for filter paper, storing a separation medium and a transfer film, and transferring 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. ,
2. A transfer method according to claim 1, wherein at least a part of the anode electrode, which is one of the pair of electrodes for flowing current, is made of iron.