JP2009063454A - Electrophoretic transfer device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrophoretic transfer device eliminating removal of a sample from a gel device when the sample is electrophoretically separated and transferred. <P>SOLUTION: The electrophoretic transfer device 10 includes a pair of electrodes 1, 2 located by holding a sample separating gel 5 between them, and an electrode moving unit 4 for moving the pair of electrodes 1, 2 to the sample separating gel 5 from a first arrangement for electrophoretically separating to a second arrangement for transferring. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an electrophoretic transfer apparatus used for electrophoresis and transfer of a sample.

  Various techniques for separating and detecting samples such as proteins and nucleic acids according to the difference in their properties have been developed, and devices for that purpose have been developed. Separation techniques include gel electrophoresis, capillary electrophoresis, liquid chromatography, and the like. Gel electrophoresis is widely used because of its simplicity and high resolution.

  Gel electrophoresis includes electrophoresis that separates a sample only in one direction and two-dimensional electrophoresis that separates a sample in two directions. Two-dimensional electrophoresis is generally performed when analyzing proteins.

  When separating proteins by two-dimensional electrophoresis, isoelectric focusing using a polyacrylamide gel is used for separation in a one-dimensional direction. Isoelectric focusing is a technique in which a voltage is applied to a gel and separation is performed based on the difference in isoelectric points of proteins. For separation in the two-dimensional direction, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using sodium anionic surfactant sodium dodecyl sulfate (SDS) is widely used. Two-dimensional electrophoresis is widely used for proteomic analysis because it can separate and analyze many proteins at once.

  In addition, as a technique for detecting a sample such as protein or nucleic acid separated by electrophoresis, transfer (blotting) is performed by moving the separated sample in the gel to the transfer film and fixing it, and the sample transferred to the film is And a method of detecting using a labeled antibody or probe. As the transfer film, a nitrocellulose film or a PVDF (Polyvinylidene difluoride) film that is easy to bind a sample and has high hydrophobicity is used. Examples of such techniques include Western blotting that detects proteins using antibodies, Northern blotting that detects RNA using labeled nucleic acid probes, and Southern blotting that detects DNA using labeled nucleic acid probes. Are known.

  In general, an electrophoresis apparatus for performing electrophoresis and a transfer apparatus for performing transfer exist as independent apparatuses. FIG. 9A is a cross-sectional view of a conventional electrophoresis apparatus, and FIG. 9B is a cross-sectional view of a general transfer apparatus.

  In the electrophoresis apparatus 80 shown in FIG. 9A, a cathode side electrode 81 and an anode side electrode 82 are fixed to a holder 85. The inside of the holder 85 is filled with the electrophoresis buffer 84. The gel 83 is fixed to the apparatus by a holder 85. Nucleic acid molecule samples such as DNA and RNA have a negative charge, and therefore, by applying a voltage from a power source (not shown) connected to the cathode side electrode 81 and the anode side electrode 82, these samples move the inside of the gel 83 to the cathode side. It moves in the direction from the electrode 81 to the anode side electrode 82, and is separated due to the difference in sample characteristics. In addition, when a protein sample is separated by SDS-PAGE, since the protein is surrounded by negatively charged SDS, the protein surrounded by the SDS is applied to the inside of the gel 83 from the cathode-side electrode 81 by application of a power supply voltage. It moves in the direction of the anode electrode 82 and is separated due to the difference in sample characteristics.

  In the transfer device 90 shown in FIG. 9B, a cathode side electrode 91 and an anode side electrode 92 are fixed to a holder 95. The inside of the holder 95 is filled with a transfer buffer 94. The gel 93 is fixed to the apparatus by a holder 95, and a transfer film 96 is disposed between the gel 93 and the anode side electrode 92. By applying a voltage from a power source (not shown), the sample moves in the gel 93 from the cathode side electrode 91 to the anode side electrode 92, reaches the transfer film 96, and is transferred to the transfer film 96.

  Conventionally, in order to transfer a sample separated by electrophoresis onto a transfer film, it is necessary to remove the gel after electrophoresis from the electrophoresis apparatus, transfer it to the transfer apparatus, and perform transfer. For this reason, the operation is time-consuming, and skill is required to remove the gel after electrophoresis from the electrophoresis apparatus and move the gel to the transfer apparatus without damaging the gel.

  As a technique for solving the above-described problem, for example, a technique for performing electrical transfer using an electrophoresis apparatus is disclosed in Patent Document 1.

FIG. 10 is a diagram illustrating a configuration of the electrophoresis apparatus disclosed in Patent Document 1. In FIG. In the electrophoresis device 79 disclosed in Patent Document 1, a gel 73 is placed on a convex platform 72 of a horizontal submarine electrophoresis device 79, and a buffer solution for electrophoresis is placed inside the electrophoresis tank 71. Electrophoresis is performed in a state in which 74 is placed and the gel 73 is immersed. When the electrophoresis is completed, the gel 73 is removed from the platform 72, the film (transfer film) 78 is brought into close contact, and these are sandwiched between the plate electrodes 77 for electrical transfer and placed on the platform 72. Then, electrical transfer is performed without immersing the gel 73 and the film 78 in the buffer solution 74. Thereby, electrophoresis and transfer can be performed with one apparatus.
JP 2000-28578 A (published January 28, 2000)

  As described above, in general, during the period from electrophoresis to transfer, “set gel to electrophoresis device”, “electrophoresis”, “extraction of gel”, “gel and transfer film to transfer device” Six steps of “setting”, “transfer”, and “removing the transfer film” are necessary. Among these steps, the operation after removing the gel after electrophoresis from the electrophoresis apparatus and setting it on the transfer apparatus is performed manually. However, since the gel is highly flexible, a series of operations for removing the gel from the electrophoresis apparatus and setting it on the transfer apparatus requires the skill of an experimenter. The skill level greatly affects the reproducibility of the experiment and the accuracy of the experiment. For example, if the skill level of the experimenter is low, in the process of removing the gel after electrophoresis from the electrophoresis apparatus and setting it on the transfer apparatus, the gel is broken or bubbles are formed between the gel and the transfer film. There was a problem that good experimental results could not be obtained due to entering or distortion of the gel. Furthermore, if the gel after electrophoresis is damaged and cannot be used for transfer, an expensive and valuable sample is lost. In addition, the electrophoresis must be performed again, which increases labor.

  In the technique disclosed in Patent Document 1, transfer can be performed using an electrophoresis apparatus. However, after electrophoresis, the gel 73 shown in FIG. After being sandwiched between the plate electrodes 77 for automatic transfer, it is necessary to place them on the platform 72 again. Therefore, the skill of the experimenter is still necessary.

  Accordingly, the present invention has been made in view of the above-described problems, and an object of the present invention is to provide an electrophoretic transfer apparatus that can perform transfer using the same apparatus without removing the gel after electrophoresis from the apparatus. It is to provide.

  In order to solve the above-described problems, the electrophoretic transfer apparatus according to the present invention performs sample separation using a sample separation gel, and transfers the separated sample from the sample separation gel to a transfer object. An electrophoretic transfer apparatus, comprising: a pair of electrodes arranged with the sample separation gel sandwiched therebetween; and the arrangement of the pair of electrodes with respect to the sample separation gel; a first arrangement for performing the electrophoretic separation; An electrode moving means for changing to a second arrangement for performing transfer is provided.

  According to the above configuration, when the sample is subjected to electrophoretic separation, the electrode moving means moves the pair of electrodes arranged with the sample separation gel between them to the first arrangement for performing electrophoretic separation, thereby performing electrophoretic separation. To do. When the electrophoretic separation is completed, the pair of electrodes in the first arrangement are moved to the second arrangement for transfer by the electrode moving means while the sample separation gel is still placed in the electrophoretic transfer device. Let By bringing the transfer object into close contact with the sample separation gel in advance, the transfer can be performed after the electrode is moved to the second arrangement. That is, after the sample is electrophoretically separated, transfer to the transfer object can be performed without removing the sample separation gel containing the separated sample from the electrophoretic transfer device.

  For this reason, the experimenter has observed the occurrence of problems such as breakage and distortion of the gel and mixing of bubbles between the gel and the transfer target, which occurred when the gel after electrophoresis separation was transferred from the electrophoresis device to the transfer device. Regardless of their skill level, they can be prevented.

  Furthermore, in the electrophoretic transfer apparatus according to the present invention, in addition to the above-described configuration, the electrode moving means preferably includes a roller, and the pair of electrodes are each a film-like electrode having flexibility. It is preferable that

  According to the said structure, in addition to the effect mentioned above, since the electrode is a film | membrane shape which has a softness | flexibility, the shape of an electrode can be changed. Therefore, when it is not necessary to spread and use the film-like electrode, the area occupied by the electrode with respect to the electrophoretic transfer device can be increased by folding the film-like electrode using a plurality of rollers or winding it around the roller. Can be small. Thereby, the electrophoretic transfer device can be miniaturized.

  Furthermore, in the electrophoretic transfer device of the present invention, in addition to the above-described configuration, each of the pair of electrodes is connected via a non-conductive material, and the pair of electrodes and the non-conductive material Preferably constitutes an endless belt.

  According to the above configuration, since the pair of electrodes are connected via the non-conductive material, in addition to the above-described effect, by determining the arrangement of one electrode, the arrangement of the other electrode is also determined. Can do. Further, since the position of the pair of electrodes is changed by moving the endless belt, the number of driving devices to be used can be reduced when the electrodes are moved using the driving device.

  Furthermore, in the electrophoretic transfer device of the present invention, in addition to the above-described configuration, the pair of electrodes are preferably plate electrodes.

  According to the said structure, since an electrode is a plate-shaped electrode and is used widely, it is not necessary to produce an electrode newly. Therefore, in addition to the effects described above, the process of manufacturing the electrophoretic transfer device is simplified.

  As described above, in the electrophoretic transfer device of the present invention, the arrangement of the pair of electrodes arranged with the sample separation gel sandwiched between the first arrangement for performing electrophoretic separation and the second arrangement for performing transfer. Since the electrode moving means for changing is provided, transfer can be performed without removing the gel after electrophoretic separation of the sample from the electrophoretic transfer device, which can occur when the gel is moved from the electrophoretic device to the transfer device. It is possible to prevent gel breakage and air bubble contamination.

[Embodiment 1]
An embodiment of the electrophoretic transfer apparatus according to the present invention will be described below with reference to FIGS. 1, 2, 7, and 8. FIG.

  First, the configuration of the electrophoretic transfer apparatus according to the present embodiment will be described with reference to FIGS.

  2A is a cross-sectional view of the electrophoretic transfer device 10 along the direction of electrophoretic separation, and FIG. 2B is along the direction orthogonal to the direction of electrophoretic separation of the electrophoretic transfer device 10. FIG. As shown in FIGS. 2A and 2B, the electrophoretic transfer device 10 includes a roller (electrode moving means) 4, a pair of electrodes, that is, a cathode-side electrode 1, and an anode-side electrode inside a case 9. 2. The case 9 has a fixing portion 16 for fixing the gel (sample separation gel) 5 and the transfer film (transfer object) 7. Before starting electrophoresis, the gel (sample separation gel) 5 and the transfer film (transfer object) 7 are fixed to the fixing unit 16, and the inside of the case 9 is filled with the buffer 8.

  The cathode side electrode 1 and the anode side electrode 2 are connected to a power source (not shown) provided outside the electrophoretic transfer apparatus 10 and are used to apply a voltage to the gel 5.

  One cathode-side electrode 1 and one anode-side electrode 2 are provided for each electrophoretic transfer device 10. The cathode side electrode 1 and the anode side electrode 2 are electrodes for performing electrophoresis as well as electrodes for performing transfer. Specifically, the cathode-side electrode 1 and the anode-side electrode 2 function as electrodes for performing electrophoresis by placing the gel 5 between the gel 5 and the electrophoresis at the time of transfer. It also functions as an electrode for performing. The mechanism by which the arrangement of the cathode side electrode 1 and the anode side electrode 2 can be changed will be described later.

  The cathode side electrode 1 and the anode side electrode 2 are flexible film-like electrodes. The cathode side electrode 1 and the anode side electrode 2 are connected to each other via a non-conductive and flexible guide sheet (non-conductive material) 3. The cathode side electrode 1 and the anode side electrode 2 are connected to each other. The guide sheet 3 constitutes one loop-shaped sheet (endless belt) 17. As the guide sheet 3, a sheet made of polyimide, polyester, polycarbonate, fluororesin, silicone, vinyl chloride resin, or the like can be used.

  The sheet 17 is installed in the case 9 so that the gel 5 used for separating the sample can be disposed inside the loop. The lower surface (the surface facing the bottom of the case 9) and the upper surface of the gel 5 always face a part of the surface constituting the inner wall of the loop of the sheet 17. The gel 5 may be a conventionally used gel such as an acrylamide gel and an agarose gel, and may be prepared using a known technique. For example, a general flat gel having a width of 80 mm × vertical width of 80 mm and a thickness of 1 mm can be used. Here, the width of the gel is the length of the gel 5 that is orthogonal to the direction in which the sample is electrophoresed and is parallel to the upper surface, and the vertical width of the gel is along the direction in which the sample is electrophoresed and separated. The length of the gel 5. A larger gel 5 can also be used by increasing the size of the electrophoretic transfer device 10.

  The sizes of the cathode side electrode 1 and the anode side electrode 2 are equal, the width of each electrode is preferably larger than the width of the gel, and the vertical width of each electrode is preferably larger than the vertical width of the gel. Here, the horizontal width of the electrode is the length along the direction orthogonal to the direction in which the sample is migrated and separated, and the vertical width of the electrode is the cathode side electrode 1 or the anode side electrode 2 arranged in a plane. Is the length along the direction in which the sample is separated by electrophoresis. With such a configuration, a voltage can be applied to the entire gel during electrophoresis and transfer. Moreover, the thickness of the cathode side electrode 1 and the anode side electrode 2 can be 50 micrometers-150 micrometers, for example, However, As long as it has sufficient intensity | strength and a softness | flexibility, it is not limited to the said range. .

  As the flexible cathode side electrode 1 and anode side electrode 2, a flexible polymer sheet in which platinum is vapor-deposited, or a flexible sheet in which a conductive substance such as carbon is kneaded in a resin Can be used.

  As a method for depositing platinum on the polymer sheet, a known technique such as vacuum deposition or sputtering may be used. In this case, it is preferable to vapor-deposit so that a film thickness may be 20-200 nm. The polymer sheet has sufficient strength and flexibility and can be used without particular limitation as long as it can deposit platinum. For example, polyvinyl chloride, polystyrene, and the like can be used. Can be used. Moreover, as a flexible sheet | seat in which electroconductive substances, such as carbon, were kneaded in resin, the commercially available electroconductive sheet with which carbon material was filled into resin or rubber | gum can be used.

  In order to change the arrangement of the cathode side electrode 1 and the anode side electrode 2 at different positions as described above, in this embodiment, two rollers 4 arranged at predetermined positions of the electrophoretic transfer apparatus 10 are used.

  The two rollers 4 are arranged inside the case 9 so that the respective rotation shafts 11 are parallel to each other, and the respective rotation shafts 11 are parallel to the bottom surface of the case 9 so that the heights from the bottom surfaces are equal. Is attached. In the present embodiment, the diameters of the rollers 4 are equal to each other. In this embodiment, as shown in FIG. 2 (a), one roller 4 is disposed at each end of the case 9, but the present invention is not limited to this. The one roller 4 should just be attached. Therefore, there may be a plurality of rollers 4 attached to both ends of the case 9. The configuration in which a plurality of rollers are provided at both ends of the case 9 will be described in the second embodiment.

  The gel 5 is disposed between the two rollers 4 as will be described in detail later. Specifically, the gel 5 is formed such that a virtual surface (a surface parallel to the bottom surface of the case 9) formed by connecting the rotation shafts 11 of the two rollers 4 is parallel to the top surface of the gel 5. Is placed.

  The two rollers 4 are disposed inside the loop of the sheet 17 so that the rotation shaft 11 and the width direction of the electrodes are parallel to each other. Here, the two rollers 4 are arranged at a position where a predetermined tension can be applied to the loop-shaped sheet 17 from the inside to the outside of the loop. As a result, the sheet 17 is held inside the case 9 via the roller 4 without sagging. In order to apply an appropriate tension to the sheet 17, the mounting position of the roller 4 may be adjusted. In this way, by applying an appropriate tension to the sheet 17, it is possible to prevent the sheet 17 from sagging, and the positions of the cathode side electrode 1 and the anode side electrode 2 are in an arrangement for performing the transfer described later. In addition, the distance between the cathode side electrode 1 and the anode side electrode 2 can be kept constant.

  A motor is connected to at least one of the two rollers 4, and the roller 4 is configured to rotate around the rotation shaft 11 by driving by the motor. As the roller 4 rotates, the sheet 17 in contact with the roller 4 also rotates as described above. Thereby, the position of the cathode side electrode 1 and the anode side electrode 2 can be moved to the optimal position for electrophoresis or transfer.

  As described above, in order to change the arrangement of the cathode side electrode 1 and the anode side electrode 2 by rotating the roller 4, it is necessary to accurately transmit the rotation of the roller 4 to the sheet 17. Therefore, in this embodiment, the roller 4 uses a fluororesin core wound with silicone rubber. Since the surface of the silicone rubber is difficult to slip, the sheet 17 can be moved without sliding. That is, the rotation of the roller 4 can be accurately transmitted to the sheet 17. Further, as a mechanism for reliably moving the sheet 17, a concavo-convex structure may be provided on the surfaces of both ends of the roller 4.

  The transfer film 7 for transferring and fixing the separated sample is a conventionally used transfer film 7 such as a nylon film, a nitrocellulose film, and a PVDF film, and a film that can easily bind nucleic acid or protein should be used. Can do.

  The gel 5 and the transfer film 7 are fixed to the fixing unit 16 before electrophoresis. At this time, a porous non-conductive hard sheet can be used. In the case of using such a sheet, before placing the transfer film 7 on the fixing portion 16, a porous and non-conductive hard sheet is previously placed on the fixing portion 16, and the transfer film 7 is placed thereon. And place the gel 5 on top of it. In order to prevent the transfer film 7 from being damaged, a filter paper may be placed. When the hard sheet is not used, first, the transfer film 7 is tensioned and the transfer film 7 is fixed to the fixing portion 16 using the pins 53. Then, the gel 5 is placed on the transfer film 7. At this time, since the gel 5 is placed on the tensioned transfer film 7, the gel 5 does not bend. When the transfer film 7 is fixed using the pins 53, the gel 5 can also be fixed by piercing the gel 53 into the pins 53 as shown in FIGS. 8 (a) and 8 (b). The diameter of the pin 53 is about 1 mm, and the number used may be determined by the size of the gel 5. In the present embodiment, the gel 5 is fixed using 12 pins 53. The fixing part 16 has a structure in contact with only the edge of the transfer film 7 and the gel 5 so as not to inhibit the applied voltage during electrophoresis and transfer. Further, when the pin 53 is not used, irregularities that will prevent the gel 5 from slipping may be provided on the bottom surface and the side surface of the fixing portion 16 that contact the gel 5. In addition, in this embodiment, although the fixing | fixed part 16 is previously provided in case 9, it is not provided in case 9 and you may attach to case 9 separately.

  The fixing portion 16 is provided so that the gel 5 and the transfer film 7 are arranged inside the loop of the sheet 17 in a state where the gel 5 and the transfer film 7 are set on the fixing portion 16. The gel 5 and the transfer film 7 fixed to the fixing unit 16 do not come into contact with the sheet 17. Therefore, even if the sheet 17 is rotated by the roller 4 to change the arrangement of the cathode side electrode 1 and the anode side electrode 2, the gel 5 and the transfer film 7 do not move.

  The inside of the case 9 is filled with a buffer 8 that is generally used for electrophoresis, such as a Tris-HCl buffer solution. The liquid level of the buffer 8 is above the sheet 17. That is, the entire sheet 17 is immersed in the buffer 8.

  In addition, the following methods may be sufficient as the preparation method of the gel 5, and the fixing method to the electrophoretic transfer apparatus 10. FIG.

  A gel 5 is prepared using a frame cassette shown in FIG. 7 having an opening necessary for electrophoresis and transfer. The gel preparation solution is injected into the frame cassette 40 from the opening 42. As shown in FIG. 7 (a), the plate 41 is placed on the surface on which the electrode is disposed during transfer and one surface on which the electrode is disposed during electrophoresis so that the gel preparation solution does not leak. Cover it by sticking. After polymerization and gelation, these plates 41 are removed, and as shown in FIG. 7B, the transfer film 7 is brought into close contact with the surface on which the anode-side electrode 2 is arranged during transfer, and the entire frame cassette 40 is electrophoresed. Fix to the transfer device 10. At this time, before setting the frame cassette 40, a porous and non-conductive hard sheet may be arranged on the fixed portion 16 in advance, and the frame cassette may be arranged thereon.

  Next, the arrangement of electrodes during electrophoresis and transfer in the electrophoresis transfer apparatus 10 will be described with reference to FIGS.

  FIG. 1A is a diagram showing the arrangement (first arrangement) of the anode-side electrode 2 and the cathode-side electrode 1 with respect to the gel 5 during electrophoresis, and FIG. It is a figure which shows arrangement | positioning (2nd arrangement | positioning) of the anode side electrode 2 and the cathode side electrode.

  As shown in FIG. 1A, at the time of electrophoresis, the anode-side electrode 2 and the cathode-side electrode 1 are in positions not facing the lower surface and the upper surface of the gel 5, and the gel 5 is in contact with the anode-side electrode 2 and the cathode. It arrange | positions so that it may be located between the side electrodes 1. That is, in this state, the upper surface and the lower surface of the gel 5 are opposed to the guide sheet 3. In the guide sheet 3 disposed to face the upper surface of the gel 5, a slit 6 is provided at a position corresponding to the end of the gel 5 on the cathode side. When the sample to be separated by electrophoresis is introduced into the gel 5, the sample is introduced through the slit 6. When performing one-dimensional electrophoresis only, a sample is applied to the well portion of the gel 5 prepared using a comb through a slit 6 using a pipette or the like. In addition, when performing two-dimensional electrophoresis, the sample is separated in advance by performing IPG (immobilized pH gradient) electrophoresis using another apparatus, and the gel with the support plate containing the separated sample is slit. 6 is brought into close contact with the end surface of the gel 5 on the cathode side.

  After the introduction of the sample into the gel 5 is completed, a voltage is applied to the gel 5 to start electrophoresis. When the sample is a nucleic acid molecule such as DNA or RNA, the sample has a negative charge, and therefore moves in the gel 5 along the direction 14 from the cathode side electrode 1 to the anode side electrode 2 according to the electric field. . When the sample is a protein, the negative charge is charged in advance by SDS treatment or the like, and therefore, the sample moves along the direction 14 from the cathode side electrode 1 toward the anode side electrode 2 as in the case of the nucleic acid molecule. Since the speed at which the sample moves inside the gel 5 varies depending on the molecular weight (size) of the sample, each sample is separated by the difference in the moving speed.

  When electrophoresis is completed, the sheet 17 is rotated by rotating the roller 4 using a motor. Thereby, the arrangement of the cathode side electrode 1 and the anode side electrode 2 is changed so that the cathode side electrode 1 and the anode side electrode 2 face the upper surface and the lower surface of the gel 5, respectively, as shown in FIG. Deploy. Here, the sheet 17 is moved so that the cathode side electrode 1 is always present at the position facing the upper surface of the gel 5 and the anode side electrode 2 is always located at the position facing the lower surface of the gel 5. When the buffer 8 is exchanged from the electrophoresis buffer to the transfer buffer, it can be exchanged at this time. For example, the buffer 8 can be exchanged by, for example, providing an outlet for the buffer 8 at the bottom of the case 9, removing the buffer 8 by removing the stopper, and transferring the transfer to the case 9 before starting the transfer. This can be done by injecting the buffer 8, but is not limited to this.

  Next, a voltage is applied to the gel 5 to move the sample toward the transfer film 7 to perform transfer. The transfer film 7 is in close contact with the surface of the gel 5 facing the anode side electrode 2. The transfer film 7 is brought into close contact with the gel 5 when the gel 5 is fixed to the electrophoretic transfer device 10, that is, before the electrophoresis is performed. Therefore, it is not necessary to perform an operation of removing the gel 5 from the electrophoretic transfer device 10 after electrophoresis, bringing the transfer film 7 into close contact with the gel 5 and returning it to the electrophoretic transfer device 10 again.

  When a voltage is applied when the cathode side electrode 1 and the anode side electrode 2 are in the second arrangement shown in FIG. 1B, the sample is gelled along the direction 15 from the cathode side electrode 1 to the anode side electrode 2. Move through 5. When the sample reaches the transfer film 7, it is fixed on the transfer film 7 and the transfer is completed. When the transfer is completed, the transfer film 7 is removed from the electrophoretic transfer apparatus 10, and an experiment such as an immune reaction is performed using the transfer film 7 to which the sample has been transferred, and the sample is analyzed.

  As described above, after the gel 5 is fixed to the electrophoretic transfer device 10 before starting electrophoresis, it is not possible to remove or move the gel 5 from the electrophoretic transfer device 10 until the transfer is completed. Absent. Therefore, it is possible to prevent the gel 5 after electrophoresis from being torn or distorted before transfer. Furthermore, before the electrophoresis is performed, the gel 5 and the transfer film 7 are brought into close contact with each other, so that the operation in the buffer 8 is possible, and bubbles mixed in between the gel 5 and the transfer film 7 are removed. Easy to do. Further, even if the gel 5 and the transfer film 7 are fixed to the electrophoretic transfer device 10, even if the gel is damaged, the gel 5 and the transfer film 7 are before the electrophoresis. Therefore, it is possible to avoid wasting a sample and re-electrophoresis.

[Embodiment 2]
Another embodiment according to the present invention will be described below with reference to FIGS. In this embodiment, in order to explain the difference from the first embodiment, for the sake of convenience of explanation, members having the same functions as the members described in the first embodiment are denoted by the same member numbers, The description is omitted.

  First, the configuration of the electrophoretic transfer apparatus according to the present embodiment will be described with reference to FIG.

  FIG. 4 is a diagram showing a configuration of the electrophoretic transfer device 19, and the cathode side electrode 1 and the anode side electrode 2 are in a first arrangement for performing electrophoresis.

  Unlike the electrophoretic transfer apparatus 10 described in the first embodiment, the electrophoretic transfer apparatus 19 includes a plurality of rollers 13 disposed at both ends of the case 9, and the cathode side electrode 1, the anode side electrode 2, and the like. Are connected via a guide tape 12 instead of a guide sheet.

  The cathode side electrode 1 and the anode side electrode 2 are the same as those used in the first embodiment. The cathode side electrode 1 and the anode side electrode 2 are connected to each other by a non-conductive guide tape 12 and have a loop shape as a whole. The guide tape 12 has a width corresponding to the lateral width of the cathode side electrode 1 and the anode side electrode 2 smaller than that of the guide sheet 3 of the first embodiment, and is connected at both ends of the lateral width of the cathode side electrode 1 and the anode side electrode 2. ing. As the guide tape 12, polyimide, polyester, polycarbonate, fluororesin, silicone, vinyl chloride resin, or the like can be used.

  The cathode side electrode 1, the anode side electrode 2, and the guide tape 12 constitute a belt 27 on one loop. The belt 27 is held by a plurality of rollers (electrode moving means) 13 disposed inside the loop of the belt 27, and is attached to the case 9 via the rollers 13. Here, it is held by six rollers 13. All the rollers 13 are attached to the inside of the case 9 so that the respective rotation shafts 18 are parallel to each other and the respective rotation shafts 18 are parallel to the bottom surface of the case 9. Among the rollers 13 attached to one end of the case 9, the height 51 from the bottom surface of the case 9 and the roller 13 attached to the other end of the top in the vertical direction of the uppermost roller 13. Among them, the roller 13 is arranged so that the height 51 from the bottom surface of the case 9 at the top in the vertical direction of the uppermost roller 13 is equal. Of the rollers 13 attached to one end of the case 9, the lowest height in the vertical direction of the lowermost roller 13 is attached to the height 52 from the bottom surface of the case 9 and the other end. The roller 13 is arrange | positioned so that the height 52 from the bottom face of the case 9 of the vertical direction lowermost part of the roller 13 among the rollers 13 may become equal. In the present embodiment, the diameters of the rollers 13 are all equal.

  As described above, since there are a plurality of rollers 13 attached to one end of the electrophoretic transfer device 19, when the cathode side electrode 1 and the anode side electrode 2 are in the first arrangement, the cathode side electrode 1 and the anode side electrode 2 has a structure in which a plurality of 2 are folded back. Thereby, the width along the direction 14 of the space occupied by the cathode side electrode 1 and the anode side electrode 2 with respect to the electrophoretic transfer device 19 is reduced, and the electrophoretic transfer device 19 can be miniaturized.

  A motor is connected to at least one of the plurality of rollers 13, and the roller 13 is configured to rotate around the rotation shaft 18 by driving by the motor. Further, the belt 27 can apply a predetermined tension to the belt 27 from the inside to the outside of the loop by adjusting the position where the roller 13 is attached. As a result, sagging of the belt 27 can be prevented. For example, when the cathode side electrode 1 and the anode side electrode 2 are in a transfer arrangement described later, the distance between the cathode side electrode 1 and the anode side electrode 2 Can be kept constant.

  Next, the arrangement of the cathode side electrode 1 and the anode side electrode 2 during electrophoresis and transfer in the electrophoretic transfer device 19 will be described with reference to FIGS. 3 (a) and 3 (b).

  FIG. 3A is a diagram showing the arrangement (first arrangement) of the cathode-side electrode 1 and the anode-side electrode 2 with respect to the gel 5 at the time of electrophoresis, and FIG. 3B is the diagram with respect to the gel 5 at the time of transfer. It is a figure which shows the cathode side electrode 1 and the anode side electrode 2 arrangement | positioning (2nd arrangement | positioning).

  As shown in FIG. 3A, since there are a plurality of rollers 13 attached to one end of the electrophoretic transfer device 19, when the cathode side electrode 1 and the anode side electrode 2 are in the first arrangement, the cathode side electrode 1 and the anode side electrode 2 have a structure in which a plurality of times are folded back. Further, since the guide tape 12 is used to connect the cathode side electrode 1 and the anode side electrode 2, the upper surface of the gel 5 is not covered with a sheet or the like, and the gel 5 and the transfer film 7 are used when electrophoresis is performed. It is easy to fix to the apparatus and to introduce the sample into the gel 5.

  When the electrophoresis is completed, the cathode side electrode 1 and the anode side electrode 2 are moved by rotating the roller 13 using a motor. Thereby, the arrangement of the cathode side electrode 1 and the anode side electrode 2 is changed, and the cathode side electrode 1 and the anode side electrode 2 are arranged at positions facing the upper and lower surfaces of the gel 5 as shown in FIG. . Here, the belt 27 is moved so that the cathode side electrode 1 is always located at a position facing the upper surface of the gel 5 and the anode side electrode 2 is always located at a position facing the lower surface of the gel 5.

  Next, a voltage is applied to the gel 5 to move the sample toward the transfer film 7 to perform transfer.

  Also in the present embodiment, as in the first embodiment, the gel 5 is not removed from the electrophoretic transfer device 19 or moved until the transfer is completed after the electrophoresis is started. Therefore, it is possible to prevent the gel 5 after electrophoresis from being torn or distorted before transfer.

[Embodiment 3]
Another embodiment according to the present invention will be described below with reference to FIG. In this embodiment, in order to explain the difference from the first embodiment, for the sake of convenience of explanation, members having the same functions as the members described in the first embodiment are denoted by the same member numbers, The description is omitted.

  First, the configuration of the electrophoretic transfer apparatus according to the present embodiment will be described with reference to FIGS.

  FIG. 5A is a cross-sectional view of the electrophoretic transfer device 29 when the arrangement of the cathode-side electrode 1 and the anode-side electrode 2 with respect to the gel 5 is in an arrangement for performing electrophoresis (first arrangement). (B) is sectional drawing of the electrophoretic transfer apparatus 29 when the arrangement | positioning with respect to the gel 5 of the cathode side electrode 1 and the anode side electrode 2 exists in the arrangement | positioning (2nd arrangement | positioning) which performs transcription | transfer.

  Unlike the electrophoretic transfer apparatus 10 described in the first embodiment, in the electrophoretic transfer apparatus 29 of the present embodiment, the cathode side electrode 1 and the anode side electrode 2 are not connected. Further, the arrangement of the cathode side electrode 1 and the anode side electrode 2 is changed by winding the cathode side electrode 1 and the anode side electrode 2 around a roller or pulling them out.

  The cathode side electrode 1 and the anode side electrode 2 are the same as those used in the first embodiment.

  As shown in FIG. 5B, the cathode side electrode 1 and the anode side electrode 2 are each attached to a winding roller (electrode moving means) 21 via a non-conductive lead sheet 22. As the lead sheet 22, polyimide, polyester, polycarbonate, fluororesin, silicone, vinyl chloride, or the like can be used.

  The two winding rollers 21 are configured so that the respective rotation shafts 25 are parallel to each other, and the respective rotation shafts 25 are parallel to the bottom surface of the case 9 so that the heights from the bottom surfaces are equal. It is attached inside. In this embodiment, the diameter of the winding roller 21 is equal to each other.

  A motor is connected to the take-up roller 21, and the take-up roller 21 is configured to rotate around the rotation shaft 25 by driving by the motor. By rotating the winding roller 21, the lead sheet 22 and the cathode side electrode 1 or the anode side electrode 2 can be wound around the winding roller 21. Thereby, the area occupied by the cathode-side electrode 1 and the anode-side electrode 2 with respect to the electrophoretic transfer device 29 when the electrodes are in the first arrangement for performing electrophoresis is reduced, and the electrophoretic transfer device 29 can be downsized. it can.

  When the cathode side electrode 1 and the anode side electrode 2 are in the first arrangement, since the cathode side electrode 1 or the anode side electrode 2 and the lead sheet 22 are wound around the winding roller 21, the upper surface of the gel 5 is It is not covered with a sheet. Therefore, when performing electrophoresis, it becomes easy to fix the gel 5 and the transfer film 7 to the apparatus and to introduce the sample into the gel 5.

  The cathode side electrode 1 and the anode side electrode 2 are also connected to the lead sheet 22 on the side opposite to the side connected to the winding roller 21, and a guide (electrode moving means) 23 is attached via the lead sheet 22. It has been. The guide 23 is a member for guiding the cathode side electrode 1 and the anode side electrode 2 wound around the winding roller 21 to appropriate positions.

  A guide groove 24 is provided on the wall surface of the case 9 along the direction of migration separation, and the guide 23 is attached to the guide groove 24. The guide groove 24 in which the guide 23 for guiding the cathode side electrode 1 is attached is formed so as to be above the upper surface of the gel 5, and the guide 23 for guiding the anode side electrode 2 is attached. The guide groove 24 is formed so as to be located below the lower surface of the gel 5 and the transfer film 7. The guide groove 24 located below the lower surface of the gel 5 may be provided on the bottom surface instead of the wall surface of the case 9. Further, the structure of the guide groove 24 may be a convex structure instead of the groove.

  A guide roller 20 is provided between the end of the gel 5 and the winding roller 21 on the side close to the end. The rotation shaft 26 of the guide roller 20 is parallel to the rotation shaft 25 of the winding roller 21. The guide roller 20 is in contact with the cathode side electrode 1 and the anode side electrode 2 or the lead sheet 22, and faces the upper surface or the lower surface of the gel when the cathode side electrode 1 and the anode side electrode 2 are in the second arrangement. The cathode-side electrode 1 and the anode-side electrode 2 in the position are provided at positions that are parallel to the upper and lower surfaces of the gel. Thereby, the distance between the cathode side electrode 1 and the anode side electrode 2 can be kept constant when the cathode side electrode 1 and the anode side electrode 2 are in a transfer arrangement described later.

  After the electrophoresis is completed, the winding roller 21 is rotated using a motor. Thereby, as shown in FIG.5 (b), the cathode side electrode 1 and the anode side electrode 2 which were wound up by the winding roller 21 are pulled out. Here, the cathode side electrode 1 is always at a position facing the upper surface of the gel 5, and the anode side electrode is always pulled out to a position facing the lower surface of the gel 5. In this case, the take-up roller 21 pushes out the lead sheet 22 to pull out the cathode side electrode 1 and the anode side electrode 2. Therefore, the lead sheet 22 is preferably rigid enough to be extruded, and is preferably a sheet of PET or polyimide, for example, having a thickness of 50 μm to 100 μm. Further, as a means for pulling out the cathode side electrode 1 and the anode side electrode 2, the cathode side electrode 1 and the anode side electrode 2 may be pulled out by moving the guide 23 using a motor or manually.

  Next, a voltage is applied to the gel 5 to move the sample toward the transfer film 7 to perform transfer.

  Also in the present embodiment, as in the first embodiment, the gel 5 is not removed from the electrophoretic transfer device 29 or moved until the transfer is completed after the electrophoresis is started. Therefore, it is possible to prevent the gel 5 after electrophoresis from being torn or distorted before transfer.

[Embodiment 4]
Another embodiment according to the present invention will be described below with reference to FIG. In this embodiment, in order to explain the difference from the first embodiment, for the sake of convenience of explanation, members having the same functions as the members described in the first embodiment are denoted by the same member numbers, The description is omitted.

  First, the configuration of the electrophoretic transfer apparatus according to the present embodiment will be described with reference to FIGS.

  6A is a cross-sectional view of the electrophoretic transfer device 39 when the arrangement of the cathode-side electrode 1 and the anode-side electrode 2 with respect to the gel 5 is in an arrangement for performing electrophoresis (first arrangement). (B) is sectional drawing of the electrophoretic transfer apparatus 39 when the arrangement | positioning of the cathode side electrode 1 and the anode side electrode 2 with respect to the gel 5 exists in the arrangement | positioning (2nd arrangement | positioning) which performs transcription | transfer.

  Unlike the electrophoretic transfer apparatus 10 described in the first embodiment, in the electrophoretic transfer apparatus 39 according to the present embodiment, the electrode is a plate electrode, and the cathode side electrode 1 and the anode side electrode 2 are not connected. In the present embodiment, the arrangement of the cathode side electrode 1 and the anode side electrode 2 is changed by sliding the cathode side electrode 1 and the anode side electrode 2 respectively.

  The cathode side electrode 1 and the anode side electrode 2 are plate-like electrodes such as a platinum plate or a resin plate containing a conductive substance such as carbon, and may be those conventionally used. A guide (electrode moving means) 32 is provided on the cathode side electrode 1 and the anode side electrode 2. The guide 32 is attached so that it can move along the guide groove 31 provided on the wall surface of the holder case 30.

  A guide groove 31 is provided on the wall surface of the case 9 along the direction of migration separation, and the guide 32 is attached to the guide groove 31. The guide groove 31 to which the guide 32 for guiding the cathode side electrode 1 is attached is formed so as to be above the upper surface of the gel 5, and the guide 32 for guiding the anode side electrode 2 is attached. The guide groove 31 is formed so as to be located below the lower surface of the gel 5 and the transfer film 7. The guide groove 31 located below the lower surface of the gel 5 may be provided on the bottom surface instead of the wall surface of the case 9.

  A motor is connected to the guide 32, and the guide 32 is configured to move along the guide groove 31 when driven by the motor. As the guide 32 moves, the cathode side electrode 1 or the anode side electrode 2 also slides and moves. Thereby, the position of the cathode side electrode 1 and the anode side electrode 2 can be moved to the optimal position for electrophoresis or transfer.

  When performing electrophoresis, as shown in FIG. 6A, the respective electrodes are slid to positions where the cathode side electrode 1 and the anode side electrode 2 do not face the upper surface and the lower surface of the gel 5. In this state, the region for fixing the gel 5 is not covered with the cathode side electrode 1 and the anode side electrode 2, so that the gel 5 and the transfer film 7 can be fixed to the apparatus and the sample can be easily introduced into the gel 5. It becomes. After introducing the sample, electrophoresis is performed in this state.

  When the electrophoresis is completed, the cathode side electrode 1 and the anode side electrode 2 are slid and moved by moving the guide using a motor or manually. Here, the cathode side electrode 1 is slid to a position where the cathode side electrode 1 is always present at a position facing the upper surface of the gel 5. Similarly, the anode side electrode 2 is always located at a position facing the lower surface of the gel 5. The anode side electrode 2 is slid to such a position. Thereby, the cathode side electrode 1 and the anode side electrode 2 are arrange | positioned at the upper and lower surfaces of the gel 5, as shown in FIG.6 (b).

  Next, a voltage is applied to the gel 5 to move the sample toward the transfer film 7 to perform transfer.

  In addition, what is necessary is just to use what is used widely as a plate-shaped electrode, and it is not necessary to produce newly like the film-shaped electrode used for embodiment mentioned above. Therefore, by using plate-like electrodes as the cathode side electrode 1 and the anode side electrode 2, the electrophoretic transfer device 39 can be easily manufactured. Further, the film-like electrode used in Embodiment 1 may be attached to a plate-like member. In this case, as the plate-like member, for example, a fluororesin sheet having a thickness of about 0.5 mm to 1 mm can be used, but is not limited thereto.

  Also in the present embodiment, as in the first embodiment, the gel 5 is not removed from the electrophoretic transfer device 39 or moved until the transfer is completed after the electrophoresis is started. Therefore, it is possible to prevent the gel 5 after electrophoresis from being torn or distorted before transfer.

  The electrophoretic transfer device of the present invention can be a device in which the mechanisms of the above-described embodiments are arranged in parallel to simultaneously perform a plurality of electrophoresis and transfer. In this case, the power source can be used in common, and a motor for moving the electrodes can be used in combination.

  In the embodiment described above, the transfer film 7 is disposed in close contact with the lower surface of the gel 5, but may be disposed in close contact with the upper surface of the gel 5. In this case, in the second arrangement for transferring, the cathode side electrode 1 is arranged at a position facing the lower surface of the gel 5, and the anode side electrode 2 is arranged at a position facing the upper surface of the gel 5.

  In the embodiment described above, the roller or guide is driven using a motor, but it may be moved manually.

  In addition, this invention is not limited to embodiment mentioned above, A various change is possible in the range shown to the claim. In other words, embodiments obtained by combining technical means appropriately changed within the scope of the claims are also included in the technical scope of the present invention.

(Additional notes)
The electrophoretic transfer device of the present invention can also be described as having the following features. In other words, in an electrophoresis apparatus and a transfer (blotting) apparatus that are configured by an electrode and a case for fixing the electrode and can apply a voltage in a specific direction, the electrode for electrophoresis and the electrode for electrophoresis are moved. In other words, an electrode moving mechanism is provided, and the electrophoresis electrode is moved and used as a transfer electrode.
In the above configuration, it is preferable that the electrode for electrophoresis is formed of a flexible sheet-like electrode, and the electrode moving mechanism is a feeding mechanism including at least two or more rollers.
Furthermore, in the above configuration, it is preferable to change the position of the electrode by sliding the plate-like electrode.
Furthermore, it is preferable to provide a mechanism that can continuously perform electrophoresis and transfer by moving the position of the electrode with the same apparatus without performing the procedure of removing the gel after electrophoresis and setting the transfer film.

  The electrophoretic transfer apparatus according to the present invention is suitable for means for separating biological samples such as proteins and nucleic acids by electrophoresis, transferring the separated samples to a transfer film, and analyzing the sample using an immune reaction or the like. Can be used.

(A) is a perspective view which shows arrangement | positioning of the sheet-like electrode at the time of electrophoresis of the electrophoretic transfer apparatus in embodiment of this invention, (b) is arrangement | positioning of the sheet-like electrode at the time of transfer of (a). FIG. (A) is sectional drawing along the electrophoretic separation direction of the electrophoretic transfer apparatus in embodiment of this invention, (b) is the electrophoretic separation direction of the electrophoretic apparatus in embodiment of this invention. It is sectional drawing along the orthogonal direction. (A) is a perspective view which shows arrangement | positioning of the sheet-like electrode at the time of electrophoresis of the electrophoresis transfer apparatus in another embodiment of this invention, (b) is a sheet-like electrode at the time of transfer of (a) It is a perspective view which shows arrangement | positioning. It is sectional drawing along the direction which carries out electrophoretic separation of the electrophoretic transfer apparatus in another embodiment of this invention. (A) is sectional drawing at the time of the electrophoresis of the electrophoresis transfer apparatus in another embodiment of this invention, (b) is sectional drawing at the time of the transfer of the electrophoresis transfer apparatus of (a). (A) is sectional drawing at the time of the electrophoresis of the electrophoresis transfer apparatus in another embodiment of this invention, (b) is sectional drawing at the time of the transfer of the electrophoresis transfer apparatus of (a). (A) And (b) is the figure which showed the gel preparation instrument used for the electrophoretic transfer apparatus of this embodiment. (A) And (b) is sectional drawing of the electrophoretic transfer apparatus in embodiment of this invention. (A) is sectional drawing which shows the conventional electrophoresis apparatus, (b) is sectional drawing which shows the conventional transfer apparatus. (A) And (b) is a figure which shows the conventional electrophoretic transfer apparatus.

Explanation of symbols

1 cathode side electrode 2 anode side electrode 3 guide sheet (non-conductive material)
4 Roller (Electrode moving means)
5 Gel (sample separation gel)
6 Slit 7 Transfer film (transfer object)
8 Buffer 9 Case 10 Electrophoretic transfer device 11 Rotating shaft 12 Guide tape (non-conductive material)
13 Roller (Electrode moving means)
14 direction 15 direction 16 fixed portion 17 sheet 18 rotating shaft 19 electrophoretic transfer device 20 guide roller 21 take-up roller (electrode moving means)
22 Lead sheet 23 Guide (Electrode moving means)
24 Guide Groove 25 Rotating Shaft 26 Rotating Shaft 27 Belt 29 Electrophoretic Transfer Device 30 Holder Case 31 Guide Groove 32 Guide (Electrode Moving Means)
DESCRIPTION OF SYMBOLS 39 Electrophoretic transfer apparatus 40 Frame cassette 41 Board 42 Opening 51 Height 52 Height 53 Pin 71 Electrophoresis tank 72 Platform 73 Gel 74 Buffer 77 Electrode transfer plate electrode 78 Membrane 79 Electrophoresis apparatus 80 Electrophoresis apparatus 81 Cathode side electrode 82 Anode side electrode 83 Gel 84 Electrophoresis buffer 85 Holder 90 Transfer device 91 Cathode side electrode 92 Anode side electrode 93 Gel 94 Transfer buffer 95 Holder 96 Transfer film

Claims (5)

An electrophoretic transfer device that performs electrophoretic separation of a sample using a sample separation gel and transfers the electrophoretic separated sample from the sample separation gel to a transfer object,
A pair of electrodes arranged across the sample separation gel;
An electrode moving means for changing the arrangement of the pair of electrodes with respect to the sample separation gel into a first arrangement for performing the electrophoretic separation and a second arrangement for performing the transfer is provided. Electrophoretic transfer device.   The electrophoretic transfer device according to claim 1, wherein the electrode moving unit includes a roller.   The electrophoretic transfer device according to claim 1, wherein the pair of electrodes are film-like electrodes having flexibility. Each of the pair of electrodes is connected via a non-conductive material,
The electrophoretic transfer device according to claim 3, wherein the pair of electrodes and the non-conductive material constitute an endless belt.   The electrophoretic transfer device according to claim 1, wherein the pair of electrodes are plate electrodes.

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