JP2008298647A - Transfer device and transfer method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transfer device for efficiently transferring a sample in a gel to a membrane as a biomolecule analyzing method using electrophoresis. <P>SOLUTION: The transfer device is constituted so as to move a substance to be transferred contained in a first medium to a second medium and equipped with a voltage applying means for applying voltage to the first medium. The voltage applying means is characterized in that at least either one of the voltage applied to a certain position on the first medium and a voltage applying time is differentiated at least at either one of the certain position and another different position. For example, the transfer device 10, a first electrode 11 and a second electrode 12 are formed into a stripe form and respectively divided into a plurality of regions having different potentials. By this constitution, different voltage is applied to the first medium 14 depending on its position. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a transfer device.

  Electrophoresis is a widely used method for analyzing biomolecules. Moreover, when analyzing the result of electrophoresis, it is widely performed that a sample in a gel is transferred to a membrane to cause an antigen-antibody reaction or the like on the membrane.

As a method for transferring a sample in a gel to a membrane, there are a method using a capillary phenomenon, a method using a voltage difference, etc. (Non-patent Document 1).
Michie Oto “Q & A about electrophoresis” pages 161-163, Yodosha, 2005

  However, as a result of investigations by the present inventors, it has been found that in the conventional transfer method, when a sample in a gel is transferred to a membrane, a phenomenon occurs in which transfer efficiency is lowered for some samples.

  The present invention has been made in view of the above problems, and an object thereof is to provide a transfer apparatus in which a decrease in transfer efficiency is suppressed.

  As a result of intensive studies, the present inventors have found that there is an optimum transfer voltage according to the molecular weight of the sample to be transferred, and further, the voltage applied to the medium containing the sample is changed depending on the position. Thus, it was found that a decrease in transfer efficiency could be suppressed, and the present invention was completed.

  That is, the transfer device according to the present invention is a transfer device for moving a material to be transferred contained in the first medium to the second medium, and includes a voltage applying means for applying a voltage to the first medium. The voltage applying means is characterized in that at least one of a voltage applied to a certain position on the first medium and a time for applying the voltage is different from at least one of other positions different from the certain position.

  According to said structure, a voltage is applied to 1st media, such as an agarose gel containing the to-be-transferred substance, and this to-be-transferred substance is transcribe | transferred to 2nd media, such as a membrane. At this time, the voltage applied to the first medium varies depending on the position on the first medium. As a result, the molecular weight of each material to be transferred can be reduced with respect to the first medium in which the molecular weight of the material to be transferred is different depending on the position, such as a polyacrylamide gel in which the material to be transferred is separated in the medium by SDS-PAGE. A combined voltage can be applied.

  Here, according to the knowledge of the present inventors, there is an optimum voltage for transfer depending on the molecular weight of the material to be transferred. In other words, when a voltage away from a specific voltage is applied to the material to be transferred, the transfer efficiency is lowered. Here, according to said structure, since the voltage match | combined with the molecular weight of each to-be-transferred substance can be applied, the fall of transfer efficiency can be suppressed.

  As will be described later, the same effect can be obtained by adjusting the time for applying the voltage to the material to be transferred instead of adjusting the voltage to be applied to the material to be transferred.

  In the transfer apparatus, it is preferable that the voltage applying unit increases the voltage to be applied stepwise or continuously along a specific direction defined for the first medium.

  According to said structure, the voltage applied to a 1st medium increases toward the specific direction prescribed | regulated in a 1st medium. Therefore, for example, a polyacrylamide gel in which the transferred substance is separated in the medium by SDS-PAGE, the first medium in which the molecular weight of the transferred substance is increased in one direction. A voltage suitable for the molecular weight of each material to be transferred can be preferably applied.

  In the transfer apparatus, the voltage application unit may lengthen the voltage application time stepwise or continuously along a specific direction defined in the first medium.

  As described above, the same effect can be obtained by adjusting the time for applying the voltage to the material to be transferred instead of adjusting the voltage to be applied to the material to be transferred.

  In the transfer apparatus, it is preferable that the voltage application unit includes a first electrode and a second electrode, and the first electrode and the second electrode are each divided into a plurality of electrode regions insulated from each other. .

  According to said structure, the 1st electrode and the 2nd electrode are comprised by the several electrode area | region insulated from each other. Therefore, different potentials can be applied to the electrode regions, and the time for applying the potential can be easily changed.

  In the transfer device, the voltage applying unit includes a first electrode and a second electrode, and the first electrode and the second electrode are each divided into a plurality of electrode regions along the specific direction. preferable.

  According to said structure, the 1st electrode and the 2nd electrode are divided | segmented into the several electrode area | region insulated from each other along the said specific direction. For this reason, it is possible to easily increase the voltage applied to the first medium along the specific direction and to shorten the time for applying the voltage to the first medium along the specific direction.

  In the transfer apparatus, it is preferable that the divided shape of the electrode region of the first electrode and the divided shape of the electrode region of the second electrode are substantially the same.

  According to said structure, since the division | segmentation shape of the electrode area | region of a 1st electrode and the division | segmentation shape of the electrode area | region of a 2nd electrode are substantially the same, a voltage can be applied to a 1st medium with high precision. That is, since one electrode region on the first electrode and the electrode region on the second electrode corresponding to the electrode region have substantially the same shape, the electric field applied to the first medium due to the potential difference between both electrode regions is Since it is based on the shape of both the electrode regions, it can be easily predicted.

  In the transfer device, the electrode region is preferably linear.

  According to said structure, since the shape of the said electrode area | region is linear, a fine electric potential difference can be formed.

  In the transfer device, it is preferable that the linear electrode regions are arranged in parallel to each other perpendicular to the specific direction.

  According to said structure, an electric potential can be finely divided | segmented along the said specific direction.

  In the transfer device, the voltage application unit may apply a voltage to each of the electrode regions.

  According to the above configuration, since the voltage applying unit directly applies a potential to the electrode region, it is possible to easily apply a voltage according to the molecular weight of each transferred material.

  In the transfer apparatus, the voltage application unit includes at least one power source, and a plurality of conductive paths that conductively connect one of the power sources and the electrode regions of the plurality of electrode regions, and the conductive path. However, it may have at least two types of resistance values.

  According to the above configuration, when the resistance value of the conductive path that conductively connects the power source and each conductive region has two or more types of resistance values, two or more types of potentials are applied to each conductive region. Can be given. Therefore, it is possible to easily apply a voltage according to the molecular weight of each material to be transferred.

  In the transfer apparatus, the voltage applying unit may change the time for applying a potential to each electrode region.

  According to the above configuration, since the voltage applying unit controls the time for applying a potential to each electrode region, it is possible to easily apply a voltage to each material to be transferred in a time according to the molecular weight. Can do.

  In the transfer apparatus, the voltage applying unit includes a power source and a movable movable conductive portion that is conductively connected to the power source and the plurality of electrode regions. When the movable conductive portion moves, The time for applying a potential to the electrode regions may be changed.

  According to said structure, the said voltage application means can control the time which provides an electric potential to each said electrode area | region easily.

  In the transfer device, the movable conductive portion may be rod-shaped.

  According to said structure, the electric potential control means can control the time which provides an electric potential to each said electrode area | region easily.

  In the transfer device according to the present invention, the voltage applying unit applies a voltage to the first medium via the electric resistance layer, and the resistance value at a certain position on the electric resistance layer is different from the certain position. It may be different from the resistance value at.

  According to the above configuration, the voltage applying unit applies a voltage to the first medium via the electric resistance layer, and the electric resistance layer has a different resistance value depending on the position. Different voltages can be applied depending on the position on the first medium.

  In the transfer apparatus, the voltage applying unit applies a voltage to the first medium via an electric resistance layer having a resistance value that is distributed stepwise or continuously along a specific direction defined in the first medium. It is preferable to apply.

  According to the above configuration, since the electrical resistance layer has a resistance value that is distributed stepwise or continuously along a specific direction defined in the first medium, the voltage application unit includes The voltage applied to one medium can be easily increased stepwise or continuously along the specific direction.

  Preferably, the transfer device further includes a cooling unit for cooling the voltage application unit.

  According to the above configuration, the cooling unit can dissipate heat generated in the voltage application unit, and the temperature of the first medium and the second medium can be maintained at the optimum temperature, thereby preventing the medium and the transferred material from being denatured. be able to.

  A transfer method according to the present invention is a transfer method for transferring a material to be transferred contained in a first medium to a second medium, and includes a voltage applying step of applying a voltage to the first medium, The voltage application step is characterized in that at least one of a voltage to be applied to a certain position on the first medium and a time for applying the voltage is different from at least one of the other positions different from the certain position.

  According to said structure, since the voltage according to the molecular weight of each to-be-transferred substance can be applied similarly to the case where the transfer apparatus concerning this invention is used, the fall of transfer efficiency can be suppressed.

  If the present invention is used, an optimum transfer voltage can be applied according to the molecular weight of the sample, so that a transfer device that suppresses a decrease in transfer efficiency can be provided.

  The protein is transferred in the direction perpendicular to the migration direction after electrophoresis. Proteins after electrophoretic separation have a molecular weight of several hundred kD to several kD, and it is difficult to transfer them uniformly.

  In SDS-PAGE and agarose gel electrophoresis, proteins have a longer migration distance due to the molecular sieving effect due to the molecular sieving effect. Therefore, when a cell lysate or the like is separated by electrophoresis, proteins having the same molecular weight form bands, and appear on the gel surface as bands in descending order of molecular weight. For example, when SDS-PAGE is performed with a polyacrylamide gel having an acrylamide concentration of 12%, a protein having a molecular mass of about 200 kD to several kD can be separated and identified as a band.

  When a protein separated for each molecular weight is transferred using electrophoresis, when a protein having a low molecular weight to a medium molecular weight is a target, the transfer can be performed in a short time of 30 to 60 minutes. However, when a high molecular weight protein needs to be transferred, a transfer time of 60 minutes or more is required. Further, in order to transfer for a longer time, it is necessary to add SDS to the transfer buffer because SDS in the buffer is deficient. However, although SDS increases the transfer efficiency, it not only impairs the sharpness of the band after protein transfer, but also loses the stability of the low molecular weight protein to the transfer substrate and penetrates the transfer substrate. There is a problem.

  The transfer device according to the present invention realizes voltage conditions, transfer times, or current conditions corresponding to the molecular weights of the respective proteins in order to efficiently transfer the proteins of the respective molecular weights simultaneously from the gel to the transfer substrate.

  That is, the transfer device according to the present invention makes at least one of the voltage applied to a certain position on the first medium and the time for applying the voltage different from at least one of the other positions different from the certain position.

  In one embodiment, the transfer device according to the present invention is configured such that a voltage applied to a certain position on the first medium is changed from the certain position to another position behind the specific direction defined by the first medium. Equal to or higher than the applied voltage. That is, the transfer device increases the voltage to be applied stepwise or continuously along a specific direction defined for the first medium.

  In another embodiment, the transfer device according to the present invention is configured so that the time for applying a voltage to a certain position on the first medium is different from the certain position in the rear along the specific direction defined for the first medium. It is equal to or longer than the time for applying the voltage to the position. That is, the transfer device lengthens the time for applying the voltage stepwise or continuously along a specific direction defined for the first medium.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

(First embodiment)
FIG. 1 is a perspective view showing an outline of a transfer device 10 according to an embodiment (first embodiment) of the present invention. As shown in FIG. 1, the transfer device 10 according to the present embodiment includes stripe electrodes 11 and 12 (voltage application means) and a conductive path 13, and can hold the first medium 14 and the second medium 15. .

  The first medium 14 is not particularly limited as long as it contains a material to be transferred. For example, the first medium 14 is a gel generally used for electrophoresis, such as an agarose gel or a polyacrylamide gel, and includes the material to be transferred. Can be used suitably. The substance to be transferred is not particularly limited, but preparations from biological materials (for example, living organisms, body fluids, cell lines, tissue cultures, or tissue fragments) can be used, and more preferably polypeptides or Polynucleotides can be used. The second medium 15 is not particularly limited as long as it is made of a substance that can fix the transfer target substance, and a thin film is preferable. Specifically, as the second medium 15, 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 second medium is held in contact with the first medium. The description of the first medium and the second medium is the same for the other embodiments described below.

The stripe electrode 11 is an electrode divided into a plurality of electrode regions insulated from each other in one direction, and the shape of each electrode region is linear. The stripe electrode 12 is the same as the stripe electrode 11. The specific size of the stripe electrode can be appropriately changed according to the size of the transfer device, and can be, for example, a line width of 50 to 200 μm, a line interval of 50 to 200 μm, and a line length of 30 to 300 mm. As a material of the stripe electrode, a conductor such as platinum, copper, or zinc can be used. The stripe electrode can be produced by forming a metal film on an insulator such as a glass plate by a sputtering apparatus or the like and forming a groove on the metal film using a dicing saw or a CO ₂ laser engraving machine. A voltage is applied to the stripe electrodes 11 and 12 through the conductive path 13.

  FIG. 2 is a schematic diagram for explaining the operation of the transfer device 10. In the transfer device 10, the stripe electrodes 11 and 12 in the region where the high-molecular-weight transfer target substance exists (the left side in the figure) are conductively connected to a high-voltage power source via the conductive path 13, so The stripe electrodes 11 and 12 in the region where the transfer substance exists (right side in the figure) are conductively connected to a low voltage power source via a conductive path 13. Thereby, an optimum voltage can be applied to each material to be transferred in the first medium 14, and the material to be transferred can be efficiently transferred from the first medium 14 to the second medium 15. The voltage to be used can be appropriately set according to the molecular weight of the material to be transferred. For example, in the range of 1 to 500 V, preferably 5 to 40 V, the region to which the low molecular weight material to be transferred is present and the high molecular weight material to be transferred. What is necessary is just to make it increase in steps to the area | region which exists. Those skilled in the art can easily understand the optimum applied voltage by referring to a reference example described later.

(Second Embodiment)
A second embodiment of the present invention will be described. First, the outline of the transfer apparatus 20 according to the present embodiment is substantially the same as that of the transfer apparatus 10 shown in FIG. 1, and includes stripe electrodes 21 and 22 (voltage application means) and a conductive path 23, The second medium 25 can be held. However, the shape and function of the conductive path 23 are different from those of the conductive path 13. The conductive path 23 is made of a resistor.

  FIG. 3 is a schematic diagram for explaining the operation of the transfer device 20 according to the present embodiment. As shown in FIG. 3, the stripe electrodes 21 and 22 are conductively connected to a power source by a conductive path 23 which is a continuous resistor. The position where the conductive path 23 is conductively connected to the power source is the end of the first medium 24 on the side where the high-molecular-weight material to be transferred exists. Therefore, a potential gradient is formed so that the potential decreases from the end portion toward the opposite side, that is, the side where the low molecular weight transfer target substance exists in the first medium 24. Thereby, an optimum voltage can be applied to each material to be transferred, and the material to be transferred can be efficiently transferred from the first medium 24 to the second medium 25. Note that the voltage range used is the same as in the first embodiment. The resistance value of the conductive path 23 may be appropriately set according to the voltage to be applied. For example, a resistor having a resistance value of 10Ω to 10 kΩ / cm per unit length can be used.

(Third embodiment)
A third embodiment of the present invention will be described. First, the outline of the transfer device 30 according to the present embodiment is substantially the same as that of the transfer device 10 shown in FIG. 1, and includes stripe electrodes 31 and 32 (voltage application means) and a conductive path 33, The second medium 35 can be held. However, the shape and function of the conductive path 33 are different from those of the conductive path 13. The conductive path 33 is made of a movable rod-shaped member.

  FIG. 4 is a schematic diagram for explaining the operation of the transfer device 30 according to the present embodiment. In the transfer device 30, the stripe electrodes 31 and 32 are given a potential by contacting a movable conductive path 33. By sliding the conductive path 33, the connection with the conductive path 33 is sequentially disconnected from the end portions of the stripe electrodes 31 and 32 on the opposite side in the sliding direction, and no potential is applied. In contrast, no voltage is applied. As a result, a voltage can be applied to each material to be transferred for an optimum time, and the material to be transferred can be efficiently transferred from the first medium 24 to the second medium 25. FIG. 5 is a schematic diagram showing the course of the slide. As shown in FIG. 5, when the conductive longitude 33 is slid halfway, no potential is applied to the stripe electrodes 11 and 12 in the region (right side in the figure) where the low molecular weight transferred substance exists. No voltage is applied. That is, voltage application can be applied in an optimal time according to the molecular weight of the material to be transferred. As described above, there is an optimum transfer voltage for the material to be transferred, but the mobility of the material to be transferred is proportional to the product of the voltage to be applied and the time to apply the voltage, so the voltage application time is adjusted. Also, the mobility of the transferred material can be optimized. The voltage application time to be used may be appropriately set depending on the voltage to be applied, the material to be transferred, the composition and thickness of the gel, etc., but the low molecular weight transferred in the range of 1 minute to 3 hours, preferably 5 minutes to 60 minutes. What is necessary is just to make it increase from the area | region where a substance exists to the area | region where a high molecular weight to-be-transferred substance exists.

(Fourth embodiment)
FIG. 6 is a perspective view schematically showing the transfer device 40 according to an embodiment (fourth embodiment) of the present invention. As shown in FIG. 6, the transfer device 40 according to the present embodiment includes electrodes 41 and 43 (voltage applying means) and an electric resistance layer 42, and can hold the first medium 44 and the second medium 45. .

  The electric resistance layer 42 is a layer having a specific resistance distribution, and the resistance value varies depending on the position on the plane defined from above and below by the electrode 43 and the first medium 44. More specifically, in the electric resistance layer 42, the resistance value is low in the upper portion of the first medium 44 where the transfer material having a relatively high molecular weight exists, and the transfer material having a relatively low molecular weight is transferred to the first medium 44. In the upper part of the existing area, the resistance value is set to be high. As a result, a high voltage is applied to the region of the first medium 44 where the relatively high molecular weight material to be transferred exists, and a low voltage is applied to the region of the first medium 44 where the relatively low molecular weight material to be transferred exists. Is applied. That is, an optimum voltage can be applied to each material to be transferred. The resistance value in the electrical resistance layer 42 is not particularly limited, but may be in the range of several thousand Ω to several tens of thousands Ω.

  Various methods can be used to form the specific resistance distribution in the electric resistance layer 42. For example, a method of forming a specific resistance distribution by distributing materials having different resistance values in the electric resistance layer 42, or For example, a method of forming a specific resistance distribution by distributing resistance materials having various thicknesses in the electric resistance layer 42 can be used. Specific examples include a resin material added with a carbon material, a configuration used for a resistor of a general electronic component, and the like.

  FIG. 7 is a perspective view showing an outline of the electrical resistance layer 42 in one embodiment. As shown in FIG. 7, in one embodiment, the electric resistance layer 42 is formed by joining columnar resistors 46 arranged in the electric resistance layer 42 by an insulating or high-resistance bonding material 47. That is, in this embodiment, anisotropy exists in the resistance value between the thickness direction and the surface direction. Specifically, the electric resistance layer 42 has a resistance value defined by the columnar resistor 46 in the heat direction, and the electric resistance layer 42 surface direction has a high resistance value defined by the bonding material 47.

  The columnar resistor 46 is formed in a columnar shape having a diameter of several tens of μm to several mm, or another shape having the same cross-sectional area, and the height is determined by the specific resistance value of the material of the columnar resistor 46 and the electric resistance layer. What is necessary is just to set suitably from the specific resistance distribution which 42 should have. As described above, the columnar resistor 46 may be formed of a material having a different specific resistance value for each arrangement region, and the column cross-sectional area or arrangement density may be changed.

  FIG. 8 is a schematic diagram illustrating the configuration of the transfer device 40. In FIG. 8, the electric resistance layer 42 and the first medium 44 are conceptually divided into a plurality of minute regions. In the following description, it is approximated that current flows only inside each minute region and does not flow across a plurality of minute regions.

  The approximate value of the voltage applied to each region of the first medium 44 when the transfer voltage is applied is a value obtained by subtracting the voltage drop in each region of the electrical resistance layer 42 from the applied voltage of the electrodes 41 and 43. The voltage drop is estimated as a product of the current flowing through each region of the electrical resistance layer 42 and the resistance value (Rr) of the region. When the resistance value is large, the voltage drop in the above region becomes large, and the applied voltage to the first medium 44 is effectively reduced. Similarly, when the resistance value is small, the voltage drop in the electric resistance layer 42 is small, and the voltage applied to the first medium 44 is effectively large.

  That is, for example, a low electric field is set in the first medium 44 by setting the resistance value of the electric resistance layer 42 large in a region having a low molecular weight, and a low resistance value of the electric resistance layer 42 is set in a region having a high molecular weight. Optimal transfer conditions can be set for each region of the first medium 44 such as applying a high electric field to the one medium 44.

  In addition, there are non-uniformities such as distribution of transferred material in the first medium 44, ion distribution, pH distribution, or differences in the contact state of each layer such as the material of the first medium 44, the second medium 45, and the electrode. There is a case. Due to these non-uniformities, a substantial resistance value distribution exists for each minute region in the first medium 44. In the conventional method, when the same voltage is applied to the entire surface of the first medium 44 for transfer, overcurrent is concentrated in a region where the resistance value (Rg) in the surface of the first medium 44 is extremely small, There was a possibility that good transfer of the entire surface of the medium 44 could not be performed.

  On the other hand, in the transfer device 40 according to the present embodiment, since the local overcurrent is limited by the electric resistance layer 42, it is possible to transfer the entire gel surface satisfactorily. That is, when the amount of current locally increases, the voltage drop in the corresponding region of the electrical resistance layer 42 increases, and the voltage applied to the first medium 44 effectively decreases, so that overcurrent is suppressed. .

  In addition, when polyacrylamide gel or the like is used as the first medium, Rg is 50 to 500Ω in total for the entire first medium 44. At this time, Rr is a total of several electric resistance layers. In the case of about 1,000 Ω to several tens of thousands Ω, the current flowing through each region substantially depends on Rr. Therefore, overcurrent caused by nonuniform resistance distribution in the first medium 44 is suppressed.

  As described above, the transfer device 40 including the electric resistance layer 42 according to the present embodiment has the optimum transfer condition for each region due to the difference in the molecular weight of the transfer target material developed and distributed in the first medium 44. In addition to being settable, deviation from the optimum transfer condition value due to local overcurrent or the like can be suppressed, and transfer efficiency can be improved.

  Further, the transfer device 40 according to the present embodiment may further include a cooling unit for cooling the electrode 41 or 43. As a result, heat generation or the like can be dissipated, the temperature of the first medium 44 and the second medium 45 can be maintained at the optimum temperature, and denaturation of the medium and the material to be transferred can be prevented.

  As the cooling means, it is possible to use a combination of heat radiating means or cooling means such as radiating fins, heat pipes, Peltier elements, water cooling blocks, cooling parts using refrigerant, air cooling fans, or the like.

  FIG. 6 shows the transfer device 40 in the case of using heat radiating fins as cooling means. In FIG. 6, from the upper surface side, it is a structure of radiation fin-anode side electrode-electric resistance layer 42-second medium 45-first medium 44-cathode side electrode. Side) Peltier element (cooling side)-anode side electrode-electric resistance layer 42-second medium 45-first medium 44-cathode side electrode; heat dissipation fin-(heat dissipation side) Peltier element (cooling side)-cathode side electrode- Electrical resistance layer 42-first medium 44-second medium 45-anode side electrode; or radiation fin-cathode side electrode-electric resistance layer 42-first medium 44-second medium 45-anode side electrode. Good. Further, the heat radiating means and the cooling means may be configured on both the upper side and the lower side of the first medium 44 or only on one side.

  In FIG. 6, the arrangement is such that the first medium 44 is horizontal, but the apparatus may be configured so that the first medium 4 is vertical for the purpose of improving the cooling efficiency of the front and back surfaces uniformly. . Further, the electric resistance layer 42 may be configured not only on one side of the first medium 44 but also on both sides of the first medium 44. The cooling means such as the heat dissipating fins arranged adjacent to each other and the electrodes may be constituted by common parts such as the same metal body.

  The heat radiating means and the cooling means radiate heat generated by the current flowing through the electric resistance layer 42, heat generated by the current flowing through the first medium 44 and the first medium 45, and the like. Used to maintain temperature at optimum temperature.

  For example, when it is determined that the temperature rise of the first medium 44 or the first medium 45 is below an allowable range due to a small amount of current at the time of transfer or the like, it is not necessary to use a heat radiating unit or a cooling unit. Good.

  In addition, the electrical resistance layer 42 may be provided as an integral structure with the electrode 41 or 43, or may be provided as a separate member as an electrical resistance sheet.

  Further, the cooling means can be easily provided in a transfer device according to another embodiment.

(Fifth embodiment)
FIG. 9 is a perspective view schematically showing a transfer device 50 according to one embodiment (fifth embodiment) of the present invention. As shown in FIG. 9, the transfer device 50 according to the present embodiment includes plate-like electrodes 51 and 52 (voltage application means), and can hold the first medium 53 and the second medium 54.

  As shown in FIG. 9, the plate-like electrode 51 is composed of a plurality of electrode regions, and a potential is applied to each electrode region, and a voltage is applied to the first medium 53 and the second medium 54 between the upper and lower electrode regions. Apply. The potential difference between the electrode regions is large between the electrode regions sandwiching the region where the relatively high molecular weight material to be transferred exists in the first medium 53, and the region where the relatively low molecular weight material to be transferred exists in the first medium 53. Is provided so as to be small between the electrode regions sandwiching. As a method of applying a specific potential to a specific electrode region of the plate electrode 51 or 52, a well-known and conventional technique may be used. Thereby, an optimum voltage can be applied to each material to be transferred, and the material to be transferred can be efficiently transferred from the first medium 53 to the second medium 54.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and the embodiments can be 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 an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

[Reference Example 1: Examination of protein transcription efficiency]
It was investigated how the amount of transcription changes when proteins having different molecular weights are transcribed at 10V, 20V, or 40V. Using a commercially available SDS-PAGE gel (ATTO Co.), SeeBlue M.M. W. And Dylight M. et al. W. Each was applied with 5 μl and subjected to SDS-PAGE, and further transferred using a commercially available transfer apparatus under a voltage condition of 10 V, 20 V or 40 V by a standard method.

  As a result of using 25 mM Tris, 192 mM Glycine, and 20% Methaneol as the transcription buffer containing 0.1% SDS, the intensity of the 200 k-64 k protein band gradually increased to 40V. On the other hand, it has been clarified that the protein having a molecular weight of 50k or less has a smaller molecular weight, and the band intensity decreases at 20V, resulting in a decrease in the transcription amount. In addition, several k proteins could not be detected when transcribed at 40 V (FIG. 10).

  The transfer buffer of 0.1% SDS contains high transfer efficiency of high molecular weight protein, but low molecular weight protein is not adsorbed to the transfer film, and it is assumed that it penetrates or diffuses on the transfer film. . Therefore, the same transfer efficiency was examined in the hope that it would not penetrate the transfer membrane of low molecular weight protein when a transfer buffer without SDS was used.

[Reference Example 2: Examination of optimum voltage range according to molecular weight]
Seed Blue M. was isolated using an SDS-PAGE apparatus (Ato). W. And Dylight M. et al. W. Was transferred with a transcription buffer (25 mM Tris, 192 mM Glycine, and 20% Methanol) without SDS under a voltage condition of 10 V, 20 V, 30 V, or 40 V, respectively. Also, Dylight M.M. W. For each of the above, CV (variation count) was performed 4 times. As a result, the 200 k, 116 k, 97 k and 66 k proteins increased in fluorescence as the voltage increased from 10 V to 30 V, but decreased at 30 to 40 V. The proteins of 42k, 36k, and 28k had the highest fluorescence at 10V, but decreased with increasing voltage (FIGS. 11 and 12).

  From these results, it was found that in protein transfer, the lower the molecular weight, the easier it is to penetrate or diffuse through the transfer membrane. The phenomenon also occurred using PVDF membranes with high protein adsorption, in the presence of 20% methanol and without SDS addition.

〔Example〕
Based on the knowledge obtained from Reference Examples 1 and 2, the present inventors have designed and implemented a transfer apparatus that can suitably transfer even a transfer target substance composed of a plurality of molecules having different molecular weights.

  Platinum was produced on blue plate glass (100 mm × 82 mm) with a line width of 930 μm and a space of 70 μm. Striped electrodes were connected to a LabView control power source using a copper mesh to form an electrode capable of forming a potential gradient (FIG. 13).

  A filter paper and a PVDF membrane (Immobilon-FL, Millipore) were deposited in a transfer buffer (192 mM Tris, 0.1% SDS, 5% methanol) and then overlaid on the stripe electrode as shown in FIG. A photograph is shown in FIG.

  Voltages of 40 V, 30 V, 20 V, and 10 V were sequentially applied from the high molecular weight protein side. The results are shown in FIG. Moreover, the application by the uniform voltage of 20V was performed as control. The results are shown in FIG.

  Comparing FIG. 16 and FIG. 17, in the transfer device according to the present invention, there were fewer proteins penetrating and more proteins were fixed to the PVDF membrane than the 20 V uniform voltage device.

  If the present invention is used, a transfer device with improved transfer efficiency can be provided, which is useful in the field of analytical machinery and the like.

1 is a perspective view schematically showing a transfer device according to a first embodiment of the present invention. It is a schematic diagram explaining operation | movement of the transfer apparatus which concerns on 1st Embodiment of this invention. It is a schematic diagram explaining operation | movement of the transfer apparatus which concerns on 2nd Embodiment of this invention. It is a schematic diagram explaining operation | movement of the transfer apparatus which concerns on 3rd Embodiment of this invention. It is a schematic diagram explaining operation | movement of the transfer apparatus which concerns on 3rd Embodiment of this invention. It is a perspective view which shows the outline of the transfer apparatus which concerns on 4th Embodiment of this invention. It is a perspective view which shows the outline of the electrical resistance sheet | seat which concerns on 4th Embodiment of this invention. It is a schematic diagram explaining the structure of the transfer apparatus which concerns on 4th Embodiment of this invention. It is a perspective view which shows the outline of the transfer apparatus which concerns on 5th Embodiment of this invention. It is a photograph which shows the result of the transfer using the transfer device concerning a prior art. It is a table | surface which shows the optimal transfer voltage for every molecular weight. It is a graph which shows the optimal transfer voltage for every molecular weight. 1 is a schematic diagram of a transfer apparatus according to an embodiment of the present invention. 1 is a schematic diagram of a transfer apparatus according to an embodiment of the present invention. It is a photograph of the transfer device concerning one embodiment of the present invention. It is a photograph which shows the result of the transfer using the transfer apparatus concerning one Embodiment of this invention. It is a photograph which shows the result of the transfer using the transfer device concerning a prior art.

Explanation of symbols

10 Transfer device 11, 12 Stripe electrode (voltage application means)
13 Conductive path 14 First medium 15 Second medium 20 Transfer device 21, 22 Stripe electrode (voltage applying means)
23 Conductive path 24 First medium 25 Second medium 30 Transfer device 31, 32 Stripe electrode (voltage applying means)
33 Conductive path 34 First medium 35 Second medium 40 Transfer device 41, 43 Electrode (voltage applying means)
42 electric resistance layer 44 first medium 45 second medium 46 columnar resistor 47 binder 50 transfer device 51, 52 plate electrode (voltage applying means)
53 First medium 54 Second medium

Claims (17)

A transfer device for moving a material to be transferred contained in a first medium to a second medium,
Voltage applying means for applying a voltage to the first medium,
The voltage applying means makes at least one of a voltage applied to a certain position on the first medium and a time for applying the voltage different from at least one of the other positions different from the certain position. .   The transfer apparatus according to claim 1, wherein the voltage applying unit increases the applied voltage stepwise or continuously along a specific direction defined in the first medium.   The transfer apparatus according to claim 1, wherein the voltage applying unit lengthens the time for applying the voltage stepwise or continuously along a specific direction defined for the first medium. The voltage applying means includes a first electrode and a second electrode;
4. The transfer apparatus according to claim 2, wherein the first electrode and the second electrode are each divided into a plurality of electrode regions that are insulated from each other. The voltage applying means includes a first electrode and a second electrode,
4. The transfer device according to claim 2, wherein the first electrode and the second electrode are each divided into a plurality of electrode regions along the specific direction. 5.   6. The transfer device according to claim 4, wherein the divided shape of the electrode region of the first electrode and the divided shape of the electrode region of the second electrode are substantially the same.   The transfer device according to claim 4, wherein the electrode region has a linear shape.   The transfer device according to claim 7, wherein the linear electrode regions are arranged in parallel to each other perpendicular to the specific direction.   The transfer device according to claim 4, wherein the voltage applying unit applies a voltage to each of the electrode regions. The voltage applying means includes at least one power source, and a plurality of conductive paths that conductively connect one of the power sources and the electrode regions of the plurality of electrode regions,
The transfer device according to claim 9, wherein the conductive path has at least two types of resistance values.   The transfer device according to claim 9, wherein the voltage applying unit changes a time for applying a potential to each of the electrode regions. The voltage application means includes a power source and a movable movable conductive portion that is conductively connected to the power source and the plurality of electrode regions,
12. The transfer apparatus according to claim 11, wherein the time for applying a potential to each electrode region is changed by moving the movable conductive portion.   The transfer device according to claim 12, wherein the movable conductive portion has a rod shape. The voltage applying means applies a voltage to the first medium through the electric resistance layer;
The transfer device according to claim 1, wherein a resistance value at a certain position on the electric resistance layer is different from a resistance value at another position different from the certain position.   The voltage applying means applies a voltage to the first medium through an electric resistance layer having a resistance value that is distributed stepwise or continuously along a specific direction defined in the first medium. The transfer device according to claim 14.   16. The transfer apparatus according to claim 1, further comprising a cooling unit for cooling the voltage applying unit. A transfer method for transferring a material to be transferred contained in a first medium to a second medium,
Including a voltage application step of applying a voltage to the first medium;
In the voltage applying step, at least one of a voltage applied to a certain position on the first medium and a time for applying the voltage is different from at least one of the other positions different from the certain position. .