JP4362660B2 - Electrophoresis and transfer device, electrophoresis and transfer chip, and electrophoresis and transfer method - Google Patents

Description

  The present invention relates to an apparatus that serves as both an electrophoresis apparatus and a transfer apparatus.

  In proteomic analysis, primary electrophoresis, secondary electrophoresis, and transfer of the sample to the membrane are performed continuously. There is a need for a technique that simplifies these procedures.

  Patent Document 1 discloses an apparatus for automating primary electrophoresis and secondary electrophoresis.

  Patent Document 2 discloses an electrophoresis and transfer device.

  However, in the apparatus described in Patent Document 2, it is necessary to peel off the gel once after electrophoresis, which still requires a complicated and skillful operation.

Patent Document 3 proposes an electrophoresis and transfer apparatus that can be easily operated without having to peel off the gel.
JP 2007-64848 A (published March 15, 2007) JP 2000-28578 A (published January 28, 2000) JP 2006-71494 A (published March 16, 2006)

  However, there is no electrophoretic and transfer device that is practical and easy to operate.

  As a result of investigations by the present inventors, it is very difficult to perform electrophoresis in an apparatus having a configuration in which a transfer electrode is simply provided on an electrophoresis path, such as the apparatus described in Patent Document 3. there were. Specifically, in the electrophoresis and transfer apparatus configured as described above, a voltage could not be applied during electrophoresis and the sample could not be moved.

  The present invention has been made in view of the above problems, and an object thereof is to provide an electrophoresis and transfer device, an electrophoresis and transfer chip, and an electrophoresis and transfer method that are practical and easy to operate.

  As a result of intensive studies, the present inventors have determined that, even when a transfer electrode is provided on the electrophoresis path, a plurality of electrode regions insulated from each other as a transfer electrode and arranged along the electrophoresis direction are provided. The inventors have found that electrophoresis can be easily performed by using the electrode having the electrode and completed the invention.

  That is, the electrophoresis and transfer apparatus according to the present invention includes a first voltage applying means for applying a voltage in a specific direction in the first medium containing the substance to be separated, and a second in contact with the first medium in the first medium. An electrophoretic transfer device comprising a second voltage applying means for applying a voltage in a direction toward the medium, wherein the second voltage applying means are insulated from each other and arranged along the specific direction. Further, the first electrode having a plurality of electrode regions is provided.

  According to the above configuration, the electrophoresis and transfer apparatus according to the present invention can transfer the separated substance to the second medium after separating the separated substance in the first medium. For example, in the analysis of biopolymers, if the electrophoresis and transfer device is used, both the electrophoresis method and the blotting method can be easily performed, which is very useful.

  At this time, in the conventional electrophoresis and transfer apparatus, the electrode provided in the second voltage applying means for transferring the separated material in the first medium to the second medium separates the separated material in the first medium. It had a function to prevent. Therefore, it was not practical.

  However, according to said structure, the 2nd voltage application means is equipped with the 1st electrode which was insulated from each other and had the several electrode area | region arranged along the said specific direction. Since the electrode regions of the first electrode are insulated from each other and arranged along the specific direction, the separation of the substance to be separated in the first medium is not hindered. That is, according to the above configuration, electrophoresis and transfer can be performed practically and simply.

  In the electrophoresis and transfer apparatus, the electrode region is preferably linear.

  According to said structure, since the said electrode area | region is linear, when it arranges along the said specific direction, a plane is easily filled and the to-be-separated substance in a 1st medium is efficiently sent to a 2nd medium. Can be transferred.

  In the electrophoresis and transfer apparatus, it is preferable that the plurality of electrode regions are arranged in parallel to each other so as to be orthogonal to the specific direction.

  The linear electrode regions that are arranged in parallel to each other in the specific direction hardly affect the voltage applied in the specific direction, and thus preferably separates the substance to be separated in the first medium. be able to.

  It is preferable that the electrophoresis and transfer apparatus further includes a connection unit that can switch connection or non-connection to the first electrode.

  According to said structure, since the connection means can switch the connection or non-connection to a 1st electrode, it can control the electric potential of a 1st electrode easily.

  In the electrophoresis and transfer apparatus, the second voltage application unit may further include a removable second electrode.

  According to said structure, since the 2nd voltage application means is equipped with the 2nd electrode which can be removed in addition to a 1st electrode, without affecting the isolation | separation of the to-be-separated substance in a 1st medium, The substance to be separated in the first medium can be easily transferred to the second medium.

  The electrophoresis and transfer device may include a removable holder that holds the second electrode and the second medium.

  According to said structure, since a 2nd medium becomes removable, a 2nd medium can be easily used for the further analysis.

  In the electrophoresis and transfer apparatus, the second voltage applying unit may further include a second electrode having a plurality of electrode regions that are insulated from each other and arranged along the specific direction.

  According to the above configuration, the electrode regions of the second electrode are insulated from each other and arranged along the specific direction, which is the direction of separation of the substance to be separated in the first medium. Does not interfere with the separation of substances to be separated in the medium. Therefore, the material to be separated in the first medium can be easily transferred to the second medium without affecting the separation of the material to be separated in the first medium.

  An electrophoresis and transfer chip according to the present invention includes a separation part for placing a first medium containing a substance to be separated and a second medium in contact with the first medium, and a first part sandwiching the separation part. A buffer solution tank and a second buffer solution tank; and a plurality of mutually insulated electrode regions arranged on the separation unit and arranged along a direction defined by the first buffer solution tank and the second buffer solution tank. It has the 1st electrode which has it, It is characterized by the above-mentioned.

  According to the above configuration, by arranging the electrodes in the first buffer solution tank and the second buffer solution tank, a voltage is applied to the first medium placed on the separation unit, and the covering in the first medium is performed. The separation substance can be separated in the above direction. At this time, since the electrode regions of the first electrode provided on the separation part are insulated from each other and arranged along the direction, the separation of the substance to be separated is not affected.

  In addition, by using the first electrode, it is possible to apply a voltage in the direction from the first medium to the second diploid for the easily separated material to be separated, thereby realizing both electrophoresis and transfer. obtain.

  In the electrophoresis and transfer method according to the present invention, following the first voltage application step of applying a voltage in a specific direction in the first medium containing the substance to be separated, and the first voltage application step, An electrophoretic and transfer method including a second voltage applying step of applying a voltage in a direction toward the second medium in contact with the first medium, wherein the second voltage applying step is insulated from each other; and The voltage is applied using a first electrode having a plurality of electrode regions arranged along the specific direction.

  According to said structure, since the 1st electrode used in a 2nd voltage application process does not inhibit isolation | separation of the to-be-separated substance in a 1st medium in a 1st voltage application process, it can perform electrophoresis and transfer suitably. it can.

  By using the present invention, both electrophoresis and transfer can be carried out without any problem without peeling off the gel, so that an electrophoretic and transfer device that is practical and easy to operate can be provided.

[First Embodiment]
An embodiment of the present invention will be described with reference to the drawings. 1 and 2 are schematic views showing the structure of the electrophoresis and transfer apparatus 100 according to this embodiment. FIG. 1 shows the structure of the electrophoresis / transfer apparatus 100 during electrophoresis, and FIG. 2 shows the structure of the electrophoresis / transfer apparatus 100 during transfer.

  As shown in FIGS. 1 and 2, the electrophoresis and transfer apparatus 100 includes buffer baths 101 and 102, a separation unit 103, a connection unit 104, electrodes 105 and 106 (first voltage applying means), a holder 107, and stripes. An electrode 108 (second voltage application means, first electrode) is provided, and an electrode 109 (second voltage application means, second electrode), a holder 110 and a wire connection tool 111 are further added at the time of transfer. Details of the stripe electrode 108 will be described later.

  Buffer tanks 101 and 102 are used to fill the buffer. As the buffer solution, a buffer solution having a composition generally used for electrophoresis may be used. The electrode 105 is provided in the buffer solution tank 101 and has a negative potential during electrophoresis. The electrode 106 is provided in the buffer solution tank 102 and has a positive potential during electrophoresis.

  A stripe electrode 108 is provided on a region sandwiched between the buffer tanks 101 and 102. The region is divided into a separation unit 103 and a connection unit 104 by a holder 107. As shown in FIG. 1, the connection part 104 is isolated from the buffer solution tanks 101 and 102 by the holder 107, and the buffer solution is prevented from entering. In addition, the holder 107 has a structure in which an upper portion is vacant, and the stripe electrode 108 on the connection portion 104 is in a state where it can be in contact with other members.

  The first medium 120 containing the substance to be separated is placed on the separation unit 106 and held by the holder 105. Note that the holder 107 has a structure in which an upper portion is vacant, and the first medium 120 is in a state in which it can contact other members.

The substance to be separated may be any substance to be separated or analyzed by electrophoresis and transcription, and preferably a preparation from a biological material (for example, a biological individual, body fluid, cell line, tissue culture, or tissue fragment). Polypeptide or polynucleotide can be used more preferably. As the first medium 120, a gel generally used for electrophoresis, such as an agarose gel or a polyacrylamide gel, can be used. When the gel is used as the first medium, for example, the already gelled first medium 120 may be placed on the separation unit 106, or a liquid material is filled on the separation unit 106, and the material The first medium 120 may be disposed on the separation unit 106 by gelling. In addition, when the present invention is applied to an apparatus that performs electrophoresis by capillary electrophoresis, a buffer solution containing a polymer used in general capillary electrophoresis can be used as the first medium.

  At the time of transfer, the stripe electrode 108 on the connection portion 104 is connected by the connection tool 111, and a negative potential is applied. In addition, the holder 110 is disposed on the first medium 120. The holder 110 includes an electrode 109 and holds the second medium 121. The second medium 121 is in contact with the first medium 120, and the electrode 109 is disposed thereon. A positive potential is applied to the electrode 109.

  The second medium 121 is not particularly limited as long as it is made of a substance capable of fixing the substance to be separated, and a thin film is preferable. Specifically, as the second medium 121, 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 stripe electrode 108 has a structure in which linear conductors are arranged in parallel, and is arranged in a direction orthogonal to the migration direction in the electrophoresis and transfer apparatus 100. The stripe electrode 108 only needs to be made of a conductive material, and is preferably a material that does not deteriorate even when brought into contact with a buffer solution. Specifically, the stripe electrode 108 is not limited to these, but platinum, copper, zinc Etc. can be created from. The width and length of each conductor constituting the stripe electrode 108 and the distance between the conductors are not particularly limited.

  Here, the operation of the electrophoresis apparatus / transfer apparatus 100 will be described with reference to the drawings. 3 and 4 are schematic diagrams showing the operation of the electrophoresis and transfer apparatus 100. FIG. FIG. 3 shows the operation of the electrophoresis / transfer apparatus 100 during electrophoresis, and FIG. 4 shows the operation of the electrophoresis / transfer apparatus 100 during transfer.

  As shown in FIG. 3, since the electrode 105 has a negative potential and the electrode 106 has a positive potential during electrophoresis, the substance to be separated in the first medium 120 is directed from the electrode 105 toward the electrode 106. Move in the direction of the arrow in FIG. At this time, the stripe electrode 108 does not hinder the movement of the substance to be separated.

  Here, for example, as shown in the examples described later, when a flat electrode is used instead of the stripe electrode 108, the potential in the first medium 120 becomes uniform, so the substance to be separated does not move. .

  On the other hand, the stripe electrode 108 is provided so as to be orthogonal to the voltage application direction, and has a plurality of electrode regions that are insulated from each other and arranged along the application direction. Therefore, the substance to be separated in the first medium 120 can move without making the potential in the voltage application direction uniform as described above.

  As shown in FIG. 4, during transfer, the electrode 105 and the electrode 106 do not apply a voltage to the gel 120, and the second medium 121 and the electrode 109 are disposed above the first medium 120. Since a negative potential is applied to the stripe electrode 108 and a positive potential is applied to the electrode 109, the material to be separated in the first medium 120 moves from the stripe electrode 108 to the electrode 109 (second medium 121). And move. As described above, the substance to be separated can be transferred to the second medium 121.

  A known and commonly used technique may be used to switch the potentials of the electrodes 105, 106, and 109 between electrophoresis and transfer. As described above, the potential of the stripe electrode 108 can be switched by switching the contact of the connecting tool 111 with respect to the stripe electrode 108 on the connection portion 104, but is not limited thereto. Any means may be used as long as it can apply a negative potential to the stripe electrode 108. For example, the stripe electrode 108 may be mounted by a known circuit technology such as a switch.

  The voltage printed during electrophoresis and transfer may be the voltage used in a general electrophoresis apparatus or transfer apparatus, and the means for applying the voltage also uses well-known and conventional techniques other than those described above. That's fine.

  In the electrophoretic transfer device 100 according to this embodiment, the second medium 121 can be easily removed as described above. Therefore, analysis (antibody antigen reaction or the like) using the second medium 121 to which the transferred substance is transferred can be suitably performed.

[Second Embodiment]
Another embodiment of the present invention will be described with reference to the drawings. FIG. 5 is a schematic diagram showing the structure of the electrophoresis / transfer apparatus 200 according to the present embodiment.

  As shown in FIG. 5, the electrophoresis and transfer apparatus 200 includes buffer baths 201 and 202, a separation unit 203, a connection unit 204, electrodes 205 and 206 (first voltage applying means), stripe electrodes 207 and 208 (second electrodes). A voltage applying means, a first electrode and a second electrode), and a wiring component 209.

  The separation unit 203 exists in a region sandwiched between the buffer tanks 201 and 202, and holds the first medium 220 and the second medium 221 from above and below. The connection unit 204 is adjacent to the separation unit 203 outside the region sandwiched between the migration tanks 201 and 202. The stripe electrodes 207 and 208 are provided vertically from the separation unit 203 to the connection unit 204, and sandwich the first medium 220 and the second medium 221 in the separation unit 203.

  The descriptions of the buffer baths 201 and 202, the electrodes 205 and 206, the first medium 220, the second medium 221, and the stripe electrodes 207 and 208 are the same as those described in the first embodiment.

  The connection component 209 is a component for applying a potential to the stripe electrodes 207 and 208 at the time of transfer. FIG. 6 is a diagram illustrating a mechanism in which the connection component 209 applies a potential to the stripe electrodes 207 and 208. As shown in the enlarged view of the wiring component in FIG. 6, the wiring component 209 has two divided regions (shaded portions in the figure), which are not shown voltages having potentials of + and −, respectively. It is connected to supply means (power supply etc.). When the connection component 209 is rotated (in the drawing, rotating types 1 to 3) or pushed between the stripe electrodes 207 and 208 (in the drawing, taper type) in the connecting portion 204, the above-described region becomes the stripe electrodes 207 and 208. Are applied to the stripe electrodes 207 and 208, respectively, to apply a potential of + or-. Note that the method of applying a potential of + or − to the stripe electrodes 207 and 208 is not limited to the above, and may be implemented by a known circuit technique such as a switch.

  Next, the operation of the electrophoresis apparatus / transfer apparatus 200 will be described with reference to the drawings. 7 and 8 are schematic diagrams showing the operation of the electrophoresis and transfer apparatus 200. FIG. FIG. 7 shows the operation of the electrophoresis / transfer apparatus 200 during electrophoresis, and FIG. 8 shows the operation of the electrophoresis / transfer apparatus 200 during transfer.

  As shown in FIG. 7, during electrophoresis, the electrode 205 has a negative potential and the electrode 206 has a positive potential, so that the substance to be separated in the first medium 220 is directed from the electrode 205 toward the electrode 206. Move in the direction of the arrow in FIG. At this time, as described above, the stripe electrodes 207 and 208 do not hinder the movement of the substance to be separated.

  As shown in FIG. 8, at the time of transfer, the electrode 205 and the electrode 206 do not apply a voltage to the first medium 220, and a negative potential is applied to the stripe electrode 207 and a positive potential is applied to the stripe electrode 208. The substance to be separated in the medium 220 moves in the direction from the stripe electrode 207 toward the stripe electrode 208 (in the direction of the arrow in FIG. 8). As described above, the substance to be separated can be transferred to the second medium 221.

  In the above description, the case where the first medium 220 is disposed on the cathode stripe electrode 207 side and the second medium 221 is disposed on the anode stripe electrode 208 side is described.

[Third Embodiment]
The present invention also provides a fully automatic two-dimensional electrophoresis-western blotting (2DGE-WB) apparatus incorporating the above-described electrophoresis and transfer apparatus according to the present invention. FIG. 9 is a schematic diagram illustrating a structure of a 2DGE-WB apparatus 300 according to an embodiment.

  As shown in FIG. 9, the 2DGE-WB apparatus 300 according to this embodiment includes a base material 314, a sample introduction and swelling tank 301, an isoelectric focusing tank 302, an SDS equilibration tank 303, and an isoelectric focusing electrophoresis. Gel place 304, electrophoresis / transfer unit 305 (electrophoresis / transfer device), transfer stripe electrode 306 (second voltage applying means, first electrode), connection member 307, buffer baths 308 and 309, reaction bath 310, A transfer electrode 311 (second voltage applying means, second electrode) and supports 312 and 313 are provided. The isoelectric focusing gel place 304 stores an isoelectric focusing gel 320, and the electrophoresis / transfer unit 305 stores a two-dimensional electrophoresis gel 321. The support 313 is used for transfer. A transfer substrate 322 is held under the electrode 311.

  The support 312 operates as follows. First, the isoelectric focusing gel 320 is obtained from the isoelectric focusing gel storage 304 and held. Next, the isoelectric focusing gel 320 is transported to the sample introduction and swelling tank 301 to introduce and swell the sample. Then, the sample introduced into the isoelectric focusing gel 320 is subjected to isoelectric focusing in the isoelectric focusing tank 302. The isoelectric focusing gel 320 containing the separated sample is subjected to SDS equilibration in the SDS equilibration tank 303, and then transported to the electrophoresis and transfer unit 305 and brought into contact with the two-dimensional electrophoresis gel 321. . After the second-dimensional electrophoresis is performed in the electrophoresis and transfer unit 305, the support 313 arranges the transfer electrode 311 and the transfer base material 322 on the upper part of the two-dimensional electrophoresis gel 321, and the transfer stripe electrode When the connecting member 307 connects 306, the sample is transferred from the two-dimensional electrophoresis gel 321 to the transfer substrate 322. The support 313 transports the transfer substrate 322 onto which the sample has been transferred to the reaction vessel 310, and performs the antibody reaction.

  As described above, since the 2DGE-WB apparatus 300 according to the present embodiment incorporates the electrophoresis and transfer apparatus according to the present invention, the process of performing the second-dimensional electrophoresis and the process of performing the transfer are performed. Since the same member can be used, the two-dimensional electrophoresis gel 321 is not required to be transported, and the two-dimensional electrophoresis and the Western blotting can be preferably performed continuously.

[Fourth Embodiment]
The present invention further provides an electrophoresis / transfer chip that can be incorporated into the above-described electrophoresis / transfer apparatus according to the present invention. The electrophoresis and transfer chip according to the present embodiment includes a separation unit for placing a first medium containing a substance to be separated, and a first buffer solution tank and a second buffer solution tank that sandwich the separation unit. And a first electrode having a plurality of mutually insulated electrode regions arranged along the direction defined by the first buffer solution tank and the second buffer solution tank. . In the present specification, the structure including the separation unit, the first and second buffer solution tanks, and the first electrode as described above is referred to as an electrophoresis and transfer chip.

  The electrophoresis and transfer chip according to this embodiment applies a voltage to the first medium placed on the separation unit by disposing electrodes in the first buffer solution tank and the second buffer solution tank, The substance to be separated in the first medium can be separated in the above direction. At this time, the electrode regions of the first electrode provided on the separation part are arranged along the direction and insulated from each other, and thus do not affect the separation of the substance to be separated.

  Those skilled in the art will readily understand that the electrophoresis and transfer chip as described above can be easily incorporated into the electrophoresis and transfer device according to the present invention. That is, the electrophoresis and transfer chip according to this embodiment includes the separation unit, the first buffer solution tank, the second buffer solution tank, and the first electrode. By incorporating it into an apparatus equipped with an electrode for electrophoresis that can be placed in two buffer tanks and an electrode for transfer that can be placed on top of the separation section, for example, in the first embodiment described above An apparatus similar to the electrophoresis and transfer apparatus can be configured.

  Note that the electrophoresis and transfer chip according to the present embodiment may be incorporated into an electrophoresis apparatus having a first voltage application unit during electrophoresis and a transfer apparatus having a second voltage application unit during transfer. Since the electrophoresis and transfer chip can hold the first medium, it can be easily moved between the electrophoresis apparatus and the transfer apparatus.

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

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

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

[Reference Example 1: Electrophoresis without a single-sided gel plate]
In the electrophoresis and transfer apparatus according to the first embodiment described above, electrophoresis is performed in a state where there is no one gel plate. Therefore, before examining the electrophoresis and transfer apparatus according to the present invention, first, it was examined whether or not it is possible to perform electrophoresis even in the absence of one gel plate in the conventional electrophoresis apparatus.

  As shown in FIG. 10 and FIG. 11, the protein separation result was obtained even in the absence of one gel plate as in the case where both gel plates were present.

  The reason why the separated protein band is not sharp without the gel plate on one side is that the protein being separated is trapped in the polyacrylamide gel (PAGE) due to drying of the gel surface in SDS-PAGE. Can be considered. Therefore, it is preferable to adjust the moisture retention.

[Example 1: Production of stripe electrode]
Since Reference Example 1 revealed that electrophoresis could be performed without a single-sided gel plate, further investigation was advanced.

A Cr binder film was formed on a 6 cm × 5 cm glass plate having a thickness of 3 mm using a sputtering apparatus, and then about 2000 mm of Pt was formed. Next, using a dicing saw (Disco) in which a blade having a width of 100 μm was set, the Pt electrode width and the distance between the electrodes were about 100 μm and about 100 μm. Also, a striped electrode substrate having a Pt line width of 100 μm and a space between Pt lines of 100 μm using a CO ₂ laser engraving machine under the processing conditions of a laser power of 1.75 W, a processing speed of 10.8 cm / sec, and a pulse frequency of 3000 Hz A stripe electrode substrate having a Pt line width of 50 μm and a Pt line space of 50 μm could be produced (FIG. 12). In addition, the glass surface was hardly subjected to laser processing under these conditions.

[Example 2: Comparative study of substrates]
A 6 cm × 5 cm gel plate (glass plate) was assembled with a 1 mm spacer in between and placed in a gel preparation container. When a glass substrate is used as one gel plate and a stripe electrode substrate having a Pt line width of 100 μm and a Pt line width of 100 μm as the other gel plate, a substrate on which the entire substrate is formed of Pt, or A glass substrate (positive control) was used.

  Next, the prepared separation gel solution (13% acrylamide mix (acrylamide: bisacrylamide = 29.2: 0.8), 378 mM Tris-HCl (pH 8.8), 0.05% APS, 0.1% TEMED) Was added up to 7 mm from the tip, and then water was overlaid. After the separated gel solution was polymerized, water was removed, and a concentrated gel solution (4% acrylamide mix (acrylamide: bisacrylamide = 29.2: 0.8), 125 mM Tris-HCl (pH 8.8), 0.8% was prepared. 05% APS, 0.2% TEMED) was added, and then a sample comb was set. The cathode electrophoresis buffer composition was 25 mM Tris, 192 mM glycine and 0.1% SDS, and the anode electrophoresis buffer composition was 150 mM Tris-HCl (pH 8.8). The sample was mixed with a dissolved equal amount of 0.5% agarose gel, dropped into the sample well and coagulated, followed by SDS-PAGE.

  As a sample to be separated by SDS-PAGE, visible-stained SeeBlue plus 2 M.P. W. Marker (Invitrogen) and fluorescently labeled DyLight fluorescent protein molecular weight markers (PIERCE) were used.

  As a result of applying voltage at a constant current of 20 mA for 45 minutes, a clear protein band was detected even when one gel plate was a stripe electrode substrate (FIG. 13). On the other hand, it was confirmed that the protein was not moved at all on the substrate on which the entire substrate was formed of Pt, and voltage could not be applied (FIG. 14).

[Example 3: Examination of protein transfer using chip]
Using a self-made instrument in which an electrophoresis tank was separated from a gel plate imitating a chip with an empty single gel surface, it was examined that a series of steps from SDS-PAGE could be performed in one chip.

  Proteins separated by SDS-PAGE were transferred using a striped electrode substrate having a Pt line width of 100 μm and a Pt line spacing of 100 μm as a cathode and a stainless steel plate as an anode. FIG. 15 is a photograph of the chip immediately after SDS-PAGE, FIG. 16 is a photograph of the chip after transfer, and FIG. 17 is a photograph of a transfer film (Immobilon-FL, PVDF film). As shown, protein separation and transcription are performed. As described above, it was confirmed that SDS-PAGE to transfer can be performed within one chip without peeling the gel from the gel plate.

  FIG. 18 is a photograph of each process. Step 1 is when the gel is filled in the chip with stripe electrodes, Step 2 is during SDS-PAGE, Step 3 is installed in the transfer cassette (apparatus for applying voltage for transfer to the chip), and PVDF film is set Step 4 is a photograph when the anode is set, Step 5 is a time when the anode is removed after transfer, and Step 6 is a photograph when the PVDF film is removed (obtained).

[Example 4: Investigation of structure in which gel is sandwiched between stripe electrodes]
Polyacrylamide gel electrophoresis (PAGE) was performed on a chip equipped with stripe electrodes on both sides of the chip and prepared with a PVDF (polyvinylidene fluoride) membrane and gel. The influence of the presence of the stripe electrode and the presence of the PPVDF film on the electrophoresis pattern was evaluated.

  As the striped electrode, a platinum glass (50 mm × 60 mm) platinum provided by photolithography under the conditions of a line width of 50 μm, an interval of 50 μm and a repetition number of 500 times was used. As the PVDF membrane, Immobilon FL manufactured by Millipore was used. As gel formation conditions, the gel thickness was 1 mm, with the acrylamide concentration being 4% concentrated gel and 13% separation gel. As an electrophoresis sample, molecular weight marker SeeBlue Pre-stained manufactured by Invitrogen was used.

  A stripe electrode with the electrode surface facing up is placed in a polymethylmethacrylate chip substrate, and a PVDF film, 1 mm thick spacer glass, and a stripe electrode with the electrode surface facing down are stacked on top of each other and covered with a chip lid. . The acrylamide monomer solution was poured into a 1 mm thick gap to form a concentrated gel and a separation gel. Then, after introducing the sample, electrophoresis was performed under the condition of a constant voltage of 150V.

  The results are shown in FIG. As shown, a separation pattern similar to the case without the PVDF membrane could be obtained by electrophoresis using a polyacrylamide gel formed by installing a PVDF membrane on a chip equipped with stripe electrodes on both sides.

  By using the present invention, it is possible to provide an electrophoresis and transfer apparatus that is practical and easy to operate. For example, steps from SDS-PAGE to Western blotting can be performed with one chip. Furthermore, a fully automatic two-dimensional electrophoresis-Western blotting apparatus can be provided by combining with a biaxial conveyance type automation apparatus.

It is a schematic diagram which shows the structure at the time of electrophoresis of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is a schematic diagram which shows the structure at the time of the transfer of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is explanatory drawing which shows the operation | movement at the time of electrophoresis of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is explanatory drawing which shows the operation | movement at the time of the transfer of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is a schematic diagram which shows the structure of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is explanatory drawing which shows operation | movement of the wiring components of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is explanatory drawing which shows the operation | movement at the time of electrophoresis of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is explanatory drawing which shows the operation | movement at the time of the transfer of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is a schematic diagram which shows the structure of the fully automatic two-dimensional-Western blotting apparatus which concerns on one Embodiment of this invention. It is a photograph which shows the result of having performed electrophoresis in the state without one gel board. It is a photograph which shows the result of having performed electrophoresis in the state without one gel board. It is a photograph of the stripe electrode which concerns on one Embodiment of this invention. It is a photograph which shows the result of the electrophoresis of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is a graph which compares the result of electrophoresis of the electrophoresis and transfer device concerning one embodiment of the present invention, and the electrophoresis and transfer device concerning a prior art. It is the photograph immediately after electrophoresis of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention. It is the photograph after transfer of the electrophoresis and transfer device concerning one embodiment of the present invention. It is a photograph of the transfer film after transfer of the electrophoresis and transfer device concerning one embodiment of the present invention. It is a photograph of each process of electrophoresis and a transfer method concerning one embodiment of the present invention. It is a photograph which shows the result of the transfer of the electrophoresis and transfer apparatus which concerns on one Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Electrophoresis and transfer apparatus 101,102 Buffer solution tank 103 Separation part 104 Connection part 105,106 Electrode (1st voltage application means)
107 holder 108 stripe electrode (second voltage applying means, first electrode)
109 electrode (second voltage applying means, second electrode)
120 1st medium 121 2nd medium 200 Electrophoresis and transfer apparatus 201, 202 Buffer solution tank 203 Separation part 204 Connection part 205, 206 Electrode (1st voltage application means)
207 Stripe electrode (second voltage applying means, first electrode)
208 stripe electrode (second voltage applying means, second electrode)
209 Connection component 220 First medium 221 Second medium 300 Fully automatic two-dimensional electrophoresis-Western blotting apparatus 301 Sample introduction and swelling tank 302 Isoelectric focusing tank 303 SDS equilibration tank 304 Gel place for electrophoresis 305 Transfer unit 306 Transfer stripe electrode (second voltage applying means, first electrode)
307 Connection member 308, 309 Buffer tank 310 Reaction tank 311 Transfer electrode (second voltage applying means, second electrode)
312, 313 Support 320 Isoelectric focusing gel 321 Two-dimensional electrophoresis gel (first medium)
322 Transfer base material (second medium)

Claims (4)

First voltage applying means for applying a voltage in a specific direction in the first medium containing the substance to be separated;
An electrophoresis and transfer apparatus comprising: a second voltage applying unit that applies a voltage in a direction toward the second medium in contact with the first medium in the first medium,
The second voltage applying means includes a first electrode having a plurality of electrode regions insulated from each other and arranged along the specific direction ;
The second voltage applying means further comprises a removable second electrode;
An electrophoresis and transfer device comprising a removable holder for holding a second electrode and a second medium .   The electrophoresis / transfer apparatus according to claim 1, wherein the electrode region has a linear shape. The plurality of electrode regions are, electrophoresis and transfer apparatus according to claim 2, characterized in that it is arranged parallel to each other Cartesian to the particular direction.   The electrophoresis and transfer apparatus according to any one of claims 1 to 3, further comprising a connection unit that can switch connection or non-connection to the first electrode.