WO2016136079A1 - 転写膜保持器具及び分離転写装置 - Google Patents
転写膜保持器具及び分離転写装置 Download PDFInfo
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- WO2016136079A1 WO2016136079A1 PCT/JP2015/084255 JP2015084255W WO2016136079A1 WO 2016136079 A1 WO2016136079 A1 WO 2016136079A1 JP 2015084255 W JP2015084255 W JP 2015084255W WO 2016136079 A1 WO2016136079 A1 WO 2016136079A1
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- Prior art keywords
- transfer film
- transfer
- separation
- holding device
- pressing member
- Prior art date
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/4473—Arrangements for investigating the separated zones, e.g. localising zones by electric means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44739—Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
Definitions
- the present invention relates to an electrophoresis technique, and more particularly to a technique for separating a specimen by electrophoresis and transferring the separated specimen to a transfer film.
- Patent Document 1 discloses an example of an apparatus for performing discharge transfer.
- the present invention has been made in view of the above problems, and a main object of the present invention is to provide a novel technique for bringing a transfer film into close contact with a discharge portion when performing discharge transfer.
- the present inventors have conceived a novel configuration of a transfer film holding device for holding a transfer film in a separation transfer apparatus that performs discharge transfer, and completed the present invention.
- the transfer film holding device separates a specimen by electrophoresis, discharges the separated specimen from the discharge section, and moves the transfer film in contact with the discharge section.
- a transfer film holding device for holding the transfer film in a separation transfer device for transferring the specimen to the transfer film the transfer film holding device having a fixing part for fixing at least one end in the moving direction of the transfer film,
- the portion includes an elastic body that contacts the transfer film from a side opposite to the discharge section, and a pressing member that presses the transfer film against the elastic body.
- the transfer film can be suitably brought into close contact with the discharge portion when discharge transfer is performed.
- 1 is a perspective view showing a schematic configuration of a separation transfer apparatus according to an embodiment of the present invention.
- 1 is a cross-sectional view illustrating a schematic configuration of a separation transfer apparatus according to an embodiment of the present invention.
- It is sectional drawing which shows schematic structure of the transfer film holding
- It is a perspective view showing a schematic structure of a transfer film holding instrument concerning one embodiment of the present invention. It is sectional drawing for demonstrating an example of the dimension of the transfer film holding
- FIG. 1 is a perspective view schematically showing the configuration of the separation transfer apparatus 100.
- FIG. 2 is a cross-sectional view schematically showing the configuration of the separation transfer apparatus 100.
- the separation and transfer apparatus 100 separates the specimen by electrophoresis, discharges the separated specimen from the discharge section, and moves the transfer film in contact with the discharge section.
- a separation transfer apparatus for transferring the separated specimen to the transfer film a clamp (fixed portion, first fixed portion, arm portion) 20, a clamp (fixed portion, second fixed portion, arm portion) 21, a clamp Frame (arm part, connecting part) 22, carrier (arm part, part around the upper end of the side wall, second part) 23, anode buffer tank (first buffer solution tank) 30, table 31, cathode buffer tank (second buffer) Liquid tank) 40, separation unit 50, motor (drive unit) 62, ball screw (drive unit) 63, guide shaft (drive unit) 64, shaft holder (drive unit) 65, guide pole (arm unit, first part) 66 ,common And a control unit 68.
- motor drive unit
- ball screw (drive unit) 63 guide shaft (drive unit) 64, shaft holder (drive unit) 65, guide pole (arm unit, first part) 66
- the separation unit 50 accommodates a separation gel (separation medium) 52, and a first opening (discharge unit) 50 a that opens into the anode buffer tank 30 and a second opening 50 b that opens into the cathode buffer tank 40.
- the transfer film 1 is disposed so as to face the first opening 50a.
- An anode (first electrode) 32 is disposed in the anode buffer tank 30, and a cathode (second electrode) 41 is disposed in the cathode buffer tank 40.
- the cathode buffer tank 40 and the anode buffer tank 30 are filled with the buffer solution, so that the cathode 41 in the cathode buffer tank 40 and the anode 32 in the anode buffer tank 30 are in two tanks. They are electrically connected via the buffer solution, the separation gel 52, and the transfer film 1. That is, the separation transfer device 100 separates the sample introduced from the second opening 50b by the separation gel 52 by applying a voltage between the cathode 41 and the anode 32, and separates each separated component into the first opening. It is a device that is discharged from 50a and adsorbed onto the transfer film 1.
- the anode 32 is disposed in the anode buffer tank 30, and the cathode 41 is disposed in the cathode buffer tank 40.
- the anode 32 and the cathode 41 are formed from a conductive material such as metal.
- a material for forming the anode 32 and the cathode 41 for example, platinum is preferable from the viewpoint of suppressing ionization of the electrode.
- the anode 32 is arranged in the anode buffer tank 30 and the cathode 41 is not particularly limited as long as it is arranged in the cathode buffer tank 40.
- the cathode 41, the first opening 50a and the anode 32 may be arranged on a substantially straight line. If the transfer film 1 is arranged in such an arrangement as shown in FIG. 1, the lines of electric force passing through the first opening 50a are substantially perpendicular to the transfer film 1, thereby improving the accuracy of sample adsorption. obtain.
- the anode 32 is preferably arranged away from the transfer film 1. Thereby, it is possible to suppress the bubbles generated from the anode 32 from adversely affecting the adsorption of the separation component to the transfer film 1.
- the anode 32 and the cathode 41 may be used by being connected to the control unit 68 or may be used by being connected to an external power supply (DC power supply device), for example.
- an external power supply DC power supply device
- the separation transfer apparatus 100 can be started by operating the control unit 68 simultaneously with the operation of the power supply. That's fine.
- the anode buffer tank 30 and the cathode buffer tank 40 are insulating containers for retaining a buffer solution (buffer).
- the cathode buffer tank 40 is provided above the anode buffer tank 30.
- the anode buffer tank 30 is fixed on the table 31, and the cathode buffer tank 40 is fixed to the anode buffer tank 30, but the present invention is not limited to this configuration.
- the buffer solution to be put into the anode buffer tank 30 and the cathode buffer tank 40 can be any conductive buffer solution, and in particular, a buffer solution having a weakly acidic to weakly basic buffer region can be suitably used.
- buffers include Tris / glycine buffer, acetate buffer, sodium carbonate buffer, CAPS buffer, Tris / boric acid / EDTA buffer, Tris / acetic acid / EDTA buffer, MOPS, Buffers such as phosphate buffer and Tris / Tricine buffer can be used.
- the transfer film 1 is supported on the bottom of the anode buffer tank 30 from the back surface (the surface opposite to the separation portion 50) of the transfer film 1 in the movement path of the transfer film 1.
- Guides (support members) 33 and 34 are provided.
- the separation unit 50 houses a separation gel (separation medium) 52 therein.
- the separation unit 50 stands up in a substantially vertical direction, the lower part thereof is arranged in the anode buffer tank 30, and the upper part thereof is arranged so that one side thereof is in contact with the cathode buffer tank 40.
- the separation gel 52 is water-cooled by at least one of the buffer solution in the anode buffer tank 30 and the buffer solution in the cathode buffer tank 40 and can be sufficiently cooled.
- the separation unit 50 also has a first opening 50 a that opens into the anode buffer tank 30 and a second opening 50 b that opens into the cathode buffer tank 40.
- the separation gel 52 faces the anode buffer tank 30 through the first opening 50a, and faces the cathode buffer tank 40 through the second opening 50b.
- the separation unit 50 is fixed to the cathode buffer tank 40 by a lock 42 provided in the cathode buffer tank 40, but the present invention is not limited to this configuration.
- the separation unit 50 can be composed of two insulating plates 51 and 53 formed of an insulator such as glass or acrylic.
- the separation part 50 exposes the separation gel 52 by lacking a part of the insulating plate 53 in the second opening 50 b, thereby easily introducing the sample into the separation gel 52. be able to.
- the separation gel 52 is a gel for separating the sample components introduced from the second opening 50b according to the molecular weight.
- the separation gel 52 can be filled in the separation unit 50 before or after the separation unit 50 is attached to the separation transfer device 100.
- a commercially available page chip filled with the separation gel 52 may be used as the separation unit 50.
- Examples of the separation gel 52 include acrylamide gel and agarose gel.
- the lateral width of the separation gel 52 can be set to a length capable of separating a sample of 10 to 12 lanes, for example.
- a configuration in which the separation gel 52 is filled in the separation unit 50 is employed.
- a configuration in which a number of ultrafine columns called nanopillars are provided between the insulating plate 51 and the insulating plate 53 is also possible. Can be adopted.
- the first opening 50a of the separation part 50 is formed of a conductive porous material (for example, a hydrophilic PVDF (Polyvinylidene difluoride) film, a hydrophilic PTFE (Polytetrafluoroethylene) film, etc.) including the periphery thereof. It may be covered with a covering portion. Accordingly, when the transfer film 1 is in contact with or pressed against the first opening 50a (when no distance is provided between the first opening 50a and the transfer film 1), the transfer film 1 is transferred when the transfer film 1 is conveyed. The frictional resistance and damage received from the separation part 50 and the separation gel 52 can be reduced.
- a conductive porous material for example, a hydrophilic PVDF (Polyvinylidene difluoride) film, a hydrophilic PTFE (Polytetrafluoroethylene) film, etc.
- the separation part 50 stands in a substantially vertical direction, the amount of sample introduction can be increased as compared with a configuration in which the separation part 50 is installed in a substantially horizontal direction. This is because it is difficult to change the depth of the well provided in the separation gel in the horizontal electrophoresis apparatus, but the depth of the well can be easily changed in the vertical electrophoresis apparatus. This is because the amount can be easily increased.
- the transfer film 1 is preferably a sample adsorbing / holding body that allows the sample separated by the separation gel 52 to be stably stored for a long period of time and further facilitates subsequent analysis.
- the material of the transfer film 1 is preferably a material having high strength and high sample binding ability (weight that can be adsorbed per unit area).
- a PVDF film or the like is suitable when the sample is a protein.
- the PVDF membrane is preferably hydrophilized in advance using methanol or the like.
- membranes conventionally used for protein, DNA and nucleic acid adsorption such as nitrocellulose membrane or nylon membrane can also be used.
- the sample that can be separated and adsorbed in the separation transfer apparatus 100 is not limited to these, but is a preparation from a biological material (for example, a biological individual, body fluid, cell line, tissue culture, or tissue fragment), or A commercially available reagent etc. are mentioned.
- a biological material for example, a biological individual, body fluid, cell line, tissue culture, or tissue fragment
- a commercially available reagent etc. are mentioned.
- a polypeptide or polynucleotide is mentioned.
- the transfer film 1 is used in a state immersed in a buffer solution in the anode buffer tank 30.
- the transfer film 1 has a length that is used for one electrophoresis / transfer, in other words, a distance that moves within the anode buffer tank 30 in one electrophoresis / transfer. If you do.
- the transfer film 1 By configuring the transfer film 1 in this way, the operation of cutting the transfer film 1 is not required for each electrophoresis / transfer, and the usability of the separation transfer apparatus 100 can be improved.
- the lateral width of the transfer film 1 may be a length corresponding to the lateral width of the separation gel 52.
- the transfer film 1 is used while being held by an adjuster (transfer film holding device) 2.
- the adjuster 2 includes clamps 20 and 21 and a clamp frame 22, and is disposed inside the side wall of the anode buffer tank 30.
- FIG. 3A and 3B are cross-sectional views showing a schematic structure of the adjuster 2.
- FIG. 3A shows a state in which the clamps 20 and 21 do not fix the transfer film 1
- FIG. 1 shows a fixed state.
- the clamps 20 and 21 fix the end portions 1 a and 1 b in the moving direction of the transfer film 1, respectively.
- the clamp 20 includes an elastic body 20a, a pressing member 20b, and a stopper (locking member) 20c.
- the elastic body 20a is placed on the back surface (first opening 50a) of the transfer film 1.
- the transfer film 1 is fixed by pressing the transfer film 1 (the end 1a thereof) against the elastic body 20a from the surface side of the transfer film 1 by the pressing member 20b. Yes.
- the pressing member 20b is preferably configured to push the transfer film 1 into the elastic body 20a.
- the pressing member 20b is centered on an axis B that is orthogonal to the moving direction and is spaced from the transfer film 1 to the opposite side of the elastic body 20a. It is configured to rotate counterclockwise on the paper surface, and includes an abutting portion A that abuts against the transfer film 1 and presses the transfer film 1 against the elastic body 20a along with the rotation.
- the pressing member 20b for fixing the transfer film 1 is rotated in a state where the contact portion A is recessed from the elastic body 20a ((A) in FIG. 3) into the elastic body 20a (((3) in FIG. 3). B)).
- the stopper 20c is a locking member that defines the rotation range of the pressing member 20b, and the pressing member 20b does not rotate more than the state in which the contact portion A is sunk into the elastic body 20a and the transfer film 1 is fixed. So as to be locked.
- the clamp 21 includes an elastic body 21a, a pressing member 21b and a stopper (locking member) 21c.
- the transfer film 1 is pressed against the elastic body 20a from the surface side of the transfer film 1 by a pressing member 21b. It is fixed.
- the pressing member 21b is preferably configured to press the transfer film 1 into the elastic body 21a.
- the pressing member 21b is centered on an axis B that is orthogonal to the moving direction and is spaced from the transfer film 1 to the side opposite to the elastic body 21a. It is configured to rotate counterclockwise on the paper surface, and has an abutting portion A that abuts against the transfer film 1 and presses the transfer film 1 against the elastic body 21a along with the rotation.
- the pressing member 21b for fixing the transfer film 1 is rotated in a state in which the contact portion A is recessed from the elastic body 21a (FIG. 3 (A)) into the elastic body 21a (((3) of FIG. 3). B)).
- the stopper 21c is a locking member that regulates the range of rotation of the pressing member 21b, and the pressing member 21b does not rotate more than the state in which the contact portion A is fitted into the elastic body 21a and the transfer film 1 is fixed. So as to be locked.
- the pressing members 20a and 20b further include a handle portion C for the user to perform the above-described rotation operation.
- the shape of the handle portion C is not particularly limited.
- the handle portion C may be configured to extend from the shaft B in a direction different from the direction in which the contact portion A extends, and the contact portion A extends. It is preferable that the pressing members 20a and 20b extend in a direction perpendicular to the direction in which the pressing members 20a and 20b have an L-shaped shape with the axis B as a whole.
- the handle C of the pressing members 20b and 21b becomes horizontal, and the height of the pressing members 20b and 21b is reduced. can do.
- the pressing members 20b and 21b move in the anode buffer tank 30 with the transfer film 1 fixed, but the pressing members 20b and 21b at this time are moved downward to reduce the moving pressing members. It can suppress that a wave arises in an anode buffer by 20b * 21b. Thereby, better transfer can be performed.
- handle C need not be linear, and may be bent so as to be easily held by the user's hand, as shown in FIGS. 4 and 5.
- the adjuster 2 is a structure that holds the transfer film 1, and is preferably configured to maintain a state where the tension of the transfer film 1 is as high as possible. This is because if the tension of the transfer film 1 is low (the transfer film is loosened), it is difficult to make the transfer film 1 closely contact the first opening 50a even if the transfer film 1 is brought into contact with the first opening 50a of the separation unit 50. Because. As will be described below, the adjuster 2 according to the present embodiment suitably maintains the tension of the transfer film 1, and can easily bring the transfer film 1 into close contact with the first opening 50a.
- FIG. 4 is a cross-sectional view for explaining the function of the adjuster 2.
- the transfer film 1 is held by the adjuster 2, that is, when the transfer film 1 is fixed by the clamps 20 and 21, when the transfer film 1 contacts the first opening 50a of the separation unit 50, the transfer film 1 1 is pushed down by the first opening 50a, and the elastic bodies 20a and 21a are pushed down by the pushed-down transfer film 1 inside the fixed positions (X in the figure) of the clamps 20 and 21.
- the tension of the transfer film 1 can be maintained. Thereby, the transfer film 1 can be suitably adhered to the first opening 50a.
- the pressing member 20b of the clamp 20 rotates so as to drag the transfer film 1 in a direction from the end 1a of the transfer film 1 toward the end 1b.
- the transfer film 1 is pressed against the elastic body 20a. That is, when the clamp 20 is fixed, the transfer film 1 is dragged in the direction toward the inside (the direction of loosening).
- the pressing member 21b of the clamp 21 is configured to press the transfer film 1 against the elastic body 21a by rotating so as to drag the transfer film 1 in the direction from the end 1a to the end 1b of the transfer film 1. Yes. That is, when the clamp 21 is fixed, the transfer film 1 is dragged in a direction toward the outside (stretching direction).
- the transfer film 1 by first fixing the transfer film 1 with the clamp 20 and then fixing the clamp 21, a suitable tension can be successfully applied to the transfer film 1. This is because, when the clamp 20 is fixed, the transfer film 1 is not fixed by the clamp 21, so even if the transfer film 1 is dragged inward by the clamp 20, the transfer film 1 simply moves. After that, by fixing the transfer film 1 with the clamp 21 as much as possible, the transfer film 1 is further dragged outward, and the tension of the transfer film 1 can be further increased.
- the tension of the transfer film 1 can be increased because at least one of the clamps (clamp 21 in the present embodiment) is configured to drag the transfer film 1 outward during fixing. Thereby, the transfer film 1 can be suitably adhered to the first opening 50a.
- the pressing member 20b of the clamp 20 rotates so as to drag the transfer film 1 inward, and the stopper 20c is further provided so that the clamp 20 can be rotated by the stopper 20c. After the movement is locked, the transfer film 1 is prevented from shifting (loosing) inward. Thereby, when a strong force is applied to the fixed transfer film 1, it is possible to suitably prevent the transfer film 1 from being loosened.
- the contact portion A of the pressing members 20b and 21b may be provided with a non-slip member in order to prevent the transfer film 1 from being displaced during fixing.
- a non-slip member a member having a known high friction coefficient can be used.
- an anti-slip seal may be attached to the contact portion.
- anti-slip members 20d and 21d such as silicon sponge may be embedded in the contact portions A of the pressing members 20b and 21b. Thereby, it can prevent that anti-slip
- the clamp 21 may include a lock (locking member) 21e.
- the lock 21e is a locking member for preventing the pressing member 21b from returning after the pressing member 21b is rotated and the transfer film 1 is fixed. By providing the lock 21e, the lock 21e is provided on the fixed transfer film 1. When a strong force is applied, the transfer film 1 can be suitably prevented from loosening.
- the material of the elastic bodies 20a and 21a has a softness that allows the pressing members 20b and 21b and the transfer film 1 to be fitted therein, and the transfer film 1 can be fixed in pairs with the pressing members 20b and 21b.
- it will not specifically limit if it is an elastic body which can be used,
- elastomers such as silicon sponge, urethane rubber, chloroprene rubber, and fluororubber, can be used conveniently.
- the physical properties of the silicon sponge used for the elastic bodies 20a and 21a are as follows: apparent density (JIS-K6767) 0.50 g / cm 3 , tensile strength (JIS-K6251) 1.6 MPa, hardness (type E, JIS-K6253) 30).
- the physical properties of the elastic bodies 20a and 21a are not limited thereto, but the apparent density (JIS-K6767) is 0.1 to 1.0 g / cm 3 , and the tensile strength (JIS-K6251) is 0.3 to 3. It can be 0 MPa, hardness (type E, JIS-K6253) 10-100.
- FIG. 7 is a diagram illustrating an example of the dimensions of the adjuster 2.
- the distance from the axis B of the pressing member 21b to the elastic body 21a can be 8.5 mm, but is not limited thereto.
- the thickness of the elastic body 21a can be 3 mm, and the pushing distance of the elastic body 21a by the pressing member 21b can be 0.5 mm, but is not limited thereto.
- the transfer film 1 is sandwiched between the pressing members 20b and 21b, which are harder than the elastic bodies 20a and 21a, and the elastic bodies 20a and 21a.
- the transfer film 1 is sunk into the elastic bodies 20a and 21a, whereby tension can be applied to the transfer film 1.
- the transfer film 1 can be suitably adhered to the first opening 50a.
- the pressing member 21b is rotated so as to wind the transfer film 1 outward, whereby the transfer film 1 is wound and fixed. Therefore, the transfer film 1 can be fixed without sagging.
- the transfer film 1 can be fixed without slack, and the transfer film 1 is tensioned when the transfer film 1 is brought into contact with the first opening 50a.
- the close contact between the first opening 50a and the transfer film 1 becomes uniform, and the molecule (specimen) to be analyzed can be transferred to the transfer film 1 satisfactorily.
- the clamp frame 22 is a shaft member that connects the clamps 20 and 21, and connects the clamps 20 and 21 with a predetermined distance therebetween.
- the clamp frame 22 is disposed at a position sandwiching the transfer film 1 from the side in the movement direction, and thereby, the front surface (surface facing the first opening 50a) and the back surface (opposite to the first opening 50a) of the transfer film 1. It can be avoided that the clamp frame 22 overlaps the side surface. Accordingly, it is possible to prevent the clamp frame 22 from inhibiting the transfer from the separation gel 52 to the transfer film 1 and the contact of other members with the back surface of the transfer film 1 (details will be described later). Further, fixing of the transfer film 1 by the clamps 20 and 21 is not inhibited.
- the clamp frame 22 and the clamps 20 and 21 are not limited to this, but may be made of a synthetic resin such as Teflon (registered trademark), acrylic resin, or PEEK resin.
- the adjuster 2 is incorporated in the arm portion.
- the arm portion moves the transfer film 1 and brings it into contact with the first opening 50a.
- the arm portion includes the adjuster 2, the carrier 23, and the guide pole 66 that are a series of connected members.
- the guide pole 66 is a shaft member that is connected to a drive unit (shaft holder 65), which will be described later, and is disposed so as to pass outside the side wall of the anode buffer tank 30.
- the carrier 23 is a member that is connected to the guide pole 66, goes around the upper end of the side wall of the anode buffer tank 30, and is connected to the clamp 20.
- the arm portion passes from the position connected to the drive portion to the outside of the side wall of the anode buffer tank 30, wraps around the upper end of the side wall, and is connected to the inside of the side wall.
- the guide pole 66 is extended
- the carrier 23 is fitted to the guide pole 66 and extends inside the side wall across the upper end of the side wall of the anode buffer tank 30.
- the guide pole 66 is disposed outside the side wall of the anode buffer tank 30, and various operations such as removal of the anode buffer tank 30 (details will be described in Embodiment 2) and electrode setting are performed as necessary. Will not get in the way. Therefore, various operations can be successfully performed by appropriately removing the carrier 23.
- the drive unit drives the arm unit in a substantially horizontal direction, and in the present embodiment, is constituted by a motor 62, a ball screw 63, a guide shaft 64, and a shaft holder 65.
- the motor 62 rotates the ball screw 63.
- a motor whose speed can be changed may be used, or a motor having a fixed speed may be used in combination with a gear.
- the ball screw 63 penetrates the shaft holder 65 and is screwed into the shaft holder 65.
- the guide shaft 64 passes through the shaft holder 65, and the shaft holder 65 is configured to be movable along the guide shaft 64. Then, when the motor 62 rotates the ball screw 63, the shaft holder 65 is driven in the X direction (substantially horizontal direction) in the figure.
- the shaft holder 65 is connected to the arm portion (guide pole 66), and thus, the drive portion can drive the arm portion in the X direction (substantially horizontal direction) in the drawing. Since the arm portion holds the transfer film 1, the transfer film 1 moves in the X direction (substantially horizontal direction) in the figure.
- the present invention is not limited to this, and the driving unit may be configured by another driving mechanism (for example, a belt, a gear, or the like) as long as the arm unit can be driven in a substantially horizontal direction. Good.
- another driving mechanism for example, a belt, a gear, or the like
- the drive unit is provided under the anode buffer tank 30. Accordingly, it is possible to prevent the buffer solution scattered from the anode buffer tank 86 from deteriorating the durability of the drive unit and the possibility that the drive unit hinders various operations on the separation transfer apparatus 100.
- the control unit 68 is a control panel that performs various controls of the separation transfer apparatus 100 (control of the position of the arm unit, control of current and voltage applied to the anode 32 and the cathode 41, etc.).
- the control unit 68 may include a button and a switch for receiving an input from the user, a lamp for notifying the user of an operation state, a display unit, and the like.
- the guides 33 and 34 are provided at the bottom of the anode buffer tank 30 so as to support the transfer film in the moving path along which the transfer film 1 moves.
- the longitudinal directions of the guides 33 and 34 are orthogonal to the moving direction (X direction) of the transfer film 1 and are parallel to the longitudinal direction of the first opening 50a.
- the separation film 50 is bent so that the side opposite to the separation part 50 is convex when the separation part 50 (on the first opening 50a side) abuts on the surface of the transfer film 1 (separation part 50 side). It has been.
- the transfer film 1 is supported by the guides 33 and 34, and the separation unit 50 presses it and is bent so as to protrude downward (to the side opposite to the separation unit 50).
- tension is applied to the transfer film 1, and the transfer film 1 can be brought into close contact with the first opening 50a. Thereby, the transfer from the separation gel 52 to the transfer film 1 can be more suitably performed.
- the guides 33 and 34 are formed at positions sandwiching the position facing the first opening 50 a at the bottom of the anode buffer tank 30, so that the guides 33 disposed on both sides of the separation unit 50 are formed.
- the transfer film 1 is supported by 34, and the separation part 50 is pressed down and bent so as to protrude downward (opposite to the separation part).
- a uniform tension is applied to the transfer film 1, and the transfer film 1 can be evenly adhered to the first opening 50a.
- the transfer from the separation gel 52 to the transfer film 1 can be more suitably performed.
- the clamp frame 22 is disposed at a position sandwiching the transfer film 1 from the side in the movement direction, so that the guides 33 and 34 do not prevent the transfer film 1 from being supported from the back surface.
- the sample is introduced into the separation gel 52 from the second opening 50 b of the separation unit 50.
- a visible molecular weight marker for confirming the progress of electrophoresis to the sample.
- the control unit 68 controls the motor 62 to set the position of the transfer film 1 as a start position, and allows current to flow between the anode 32 and the cathode 41 to start electrophoresis.
- the value of the current that flows between the anode 32 and the cathode 41 is not particularly limited, but is preferably 50 mA or less, and more preferably 20 mA or more and 30 mA or less.
- the current value may be controlled to be constant, the voltage may be controlled to be constant, or the current / voltage may be controlled in other manners.
- the transfer film 1 is gradually moved in the X direction (substantially horizontal direction) by driving the arm unit (adjuster) by the driving unit in accordance with the progress of electrophoresis in the separation unit 50.
- the X direction is a direction orthogonal to the longitudinal direction of the first opening 50a.
- the moving speed of the transfer film 1 is not particularly limited, but can be a pace that moves 5 to 10 cm in 60 to 120 minutes, for example.
- the position of the sample discharged from the first opening 50a by electrophoresis (separated in the separation gel 52) according to the discharge timing in the transfer film 1 (the first opening 50a at the discharge timing). Adsorbed at the position facing the As a result, the separated sample is transferred to the transfer film 1.
- the transfer membrane 1 can be collected and used for staining or immune reaction (blocking and antigen-antibody reaction in Western blotting). Thereafter, a separation pattern of components transferred to the transfer film 1 is detected by a fluorescence detector or the like.
- a fluorescence detector may be incorporated in the separation and transfer apparatus 100, whereby the entire steps of electrophoresis, transfer and detection can be automated.
- the separation unit 50 is immersed in at least one buffer solution of the anode buffer tank 30 and the cathode buffer tank 40, and the separation gel 52 is water-cooled. Can do.
- the separation transfer apparatus 100 When the separation transfer apparatus 100 is configured in this way, (i) it is necessary to move the transfer film 1 in the anode buffer tank 30, and (ii) the drive unit is connected to the transfer film as in the prior art. 1, the buffer solution scattered from the anode buffer tank 30 may reduce the durability of the drive unit, and the drive unit may hinder various operations on the separation transfer apparatus 100. (Iii) In this embodiment, the drive unit is provided under the anode buffer tank 30, and the shape of the arm passes outside the side wall of the anode buffer tank 30 and wraps around the upper end of the side wall.
- the transfer film 1 is placed in the anode buffer tank 30 while avoiding a decrease in the durability of the drive unit due to the buffer solution and obstruction of various operations by the drive unit. It can be moved successfully in.
- the transfer film holding device (adjuster 2) according to aspect 1 of the present invention separates the specimen by electrophoresis, discharges the separated specimen from the discharge section (first opening 50a), and transfers the transfer film 1 to the discharge section.
- a transfer film holding device for holding the transfer film 1 in a separation transfer apparatus 100 for transferring the specimen separated by contact and movement to the transfer film, wherein at least one of the transfer films 1 is moved in the moving direction.
- a fixing portion (clamps 20 and 21) for fixing the end portion is provided.
- the fixing portion includes elastic bodies 20a and 20b that come into contact with the transfer film from the side opposite to the discharge section, and the transfer film to the elastic body. Pressing members 20b and 21b for pressing are provided.
- the elastic body comes into contact with the end portion of the transfer film from the side opposite to the discharge portion, and the end portion is fixed so that the pressing member presses the end portion against the elastic body. Yes.
- the transfer film when the transfer film is brought into contact with the discharge portion, the transfer film can be pulled into the elastic body to apply tension to the transfer film. Thereby, a transfer film can be suitably stuck to a discharge part.
- the pressing members 20b and 21b are separated from the transfer film 1 on the side opposite to the elastic bodies 20a and 21a perpendicular to the moving direction. And a contact portion A that contacts the transfer film 1 and presses the transfer film 1 against the elastic bodies 20a and 21a. Also good.
- the transfer film can be fixed by rotating the pressing member.
- the pressing members 20b and 21b may further include a handle portion C for the rotation operation.
- the pressing member can be easily rotated.
- the pressing members 20b and 21b may have an L-shape with the axis B as a curved portion.
- the pressing member can be easily rotated.
- the height of the pressing member in a state where the transfer film 1 is fixed can be reduced, it is possible to suppress waves from being generated in the anode buffer by the moving pressing member during discharge transfer, and good transfer It can be performed.
- a transfer film holding device includes the first fixing part (clamp 21) for fixing one end 1b in the moving direction of the transfer film in the above aspects 2 to 4,
- the pressing member 21b of the fixed portion rotates so as to drag the transfer film 1 in the direction from the other end 1a in the moving direction of the transfer film 1 toward the one end 1b.
- the elastic body 21a may be pressed.
- the transfer film when one end portion of the transfer film is fixed by the first fixing portion, the transfer film is dragged (rolled) outward (in the direction from the other end portion toward the one end portion).
- the pressing member is rotated. Therefore, after fixing the other end of the transfer film, by fixing the one end by the first fixing part, the transfer film is dragged outward (rolled) and fixed.
- the transfer film can be fixed without sagging.
- the first fixing portion is a locking member (stopper 21c, lock) that defines the range of rotation of the pressing member 21b of the first fixing portion. 21e) may be provided.
- the transfer film holding device includes the second fixing part (clamp 20) for fixing the other end part in the above-described aspect 5 or 6, and the pressing member 20b of the second fixing part is provided.
- the transfer film 1 may be pressed against the elastic body 20a by rotating so as to drag the transfer film 1 in the direction from the other end 1b toward the one end 1a.
- the transfer film when the other end of the transfer film is fixed by the second fixing portion, the transfer film is dragged inward (in the direction from the other end toward the one end). Even when the pressing member is rotated, when the first fixing portion fixes one end of the transfer film, the pressing member is rotated so as to slide the transfer film outward. After fixing the other end portion of the transfer film with the second fixing portion and then fixing the one end portion with the first fixing portion, the transfer film is dragged outward and fixed. Can be fixed without slack.
- the second fixing portion is a locking member (stopper 20c) that defines the range of rotation of the pressing member 20b of the second fixing portion. You may have.
- the contact portion A may be provided with anti-slip members 20d and 21d.
- the transfer film can be prevented from shifting when the transfer film is fixed.
- the anti-slip member may be embedded in the contact portion A.
- the anti-slip member can be prevented from being separated from the contact portion.
- the separation transfer apparatus separates the specimen by separating the specimen by electrophoresis, discharging the separated specimen from the discharge section, and moving the transfer film in contact with the discharge section.
- a separation transfer apparatus for transferring the transfer film to the transfer film and includes the transfer film holding device according to the first to tenth aspects.
- the separation transfer apparatus is the first transfer liquid tank according to the eleventh aspect, wherein the first buffer tank, the second buffer tank disposed above the first buffer tank, and the separation medium are accommodated.
- a separation opening that has a first opening that opens into the tank and a second opening that opens into the second buffer tank, and is positioned in a position facing the first opening;
- An arm portion to be held, and a drive portion that is provided under the first buffer solution tank and drives the arm portion in a substantially horizontal direction.
- the arm portion passes through the outside of the side wall of the first buffer solution tank, and the side wall May be connected to the inside of the side wall.
- the separation unit is configured to stand substantially vertically, so that the separation unit is immersed in the buffer solution in the first or second buffer solution tank, and the separation medium can be cooled with water.
- the separation transfer apparatus is configured in this way, it is necessary to move the transfer film in the first buffer solution tank.
- a drive part is provided under a 1st buffer solution tank, and the shape of an arm part passes along the outer side of the side wall of a 1st buffer solution tank, and wraps around the upper end of the said side wall.
- the arm portion is connected to the driving portion, and extends outside the side wall to a position aligned with the upper end of the side wall.
- the second part can be easily attached to and detached from the drive unit.
- the first part is disposed outside the side wall of the first buffer tank, and does not obstruct various operations such as removal of the first buffer tank and setting of the electrodes. Therefore, various operations can be successfully performed by appropriately removing the second part.
- the first electrode is disposed in the first buffer solution tank
- the second electrode is disposed in the second buffer solution tank
- the transfer film May be arranged so as to be inserted into the first opening and the first electrode.
- a voltage can be applied to the separation medium between the first opening opened in the first buffer solution tank and the second opening opened in the second buffer solution tank. Can be successfully performed.
- the transfer film is inserted between the first opening and the first electrode, the separated specimen can be successfully transferred from the first opening to the transfer film.
- the present invention can be used in the field of analysis of biomolecules.
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Abstract
Description
陽極32は陽極バッファ槽30内に配置されており、陰極41は陰極バッファ槽40内に配置されている。陽極32及び陰極41は、金属などの導電性を有する材料から形成される。陽極32及び陰極41を形成する材料としては、例えば電極のイオン化を抑制する観点から白金が好ましい。
陽極バッファ槽30及び陰極バッファ槽40は、緩衝液(バッファ)を滞留させる絶縁性の容器である。陰極バッファ槽40は、陽極バッファ槽30に対して上方に設けられている。なお、本実施形態では、陽極バッファ槽30はテーブル31上に固定されており、陰極バッファ槽40は陽極バッファ槽30に固定されているが、本発明はこの構成には限定されない。
分離部50は、その内部に分離ゲル(分離媒体)52を収納している。本実施形態において、分離部50は、略垂直方向に起立しており、その下部は陽極バッファ槽30内に配置され、その上部は片面が陰極バッファ槽40に接するように配置されている。これにより、分離ゲル52は、陽極バッファ槽30内の緩衝液及び陰極バッファ槽40内の緩衝液の少なくとも一方によって水冷され、十分に冷却することができる。
転写膜1は、分離ゲル52によって分離されたサンプルを長期間にわたって安定に保存可能にし、さらに、その後の分析を容易にするサンプルの吸着・保持体であることが好ましい。転写膜1の材質としては、高い強度を有し、かつサンプル結合能(単位面積当たりに吸着可能な重量)が高いものが好ましい。転写膜1としては、サンプルがタンパク質である場合にはPVDF膜などが適している。なお、PVDF膜は予めメタノールなどを用いて親水化処理を行っておくことが好ましい。これ以外には、ニトロセルロース膜又はナイロン膜など、従来からタンパク質、DNA及び核酸の吸着に利用されている膜も使用可能である。
本実施形態において、転写膜1は、アジャスタ(転写膜保持器具)2によって保持された状態で使用される。アジャスタ2は、クランプ20・21及びクランプフレーム22を備え、陽極バッファ槽30の側壁の内側に配置されている。
本実施形態において、アジャスタ2はアーム部に組み込まれている。アーム部は、転写膜1を、移動、及び、第一開口50aと当接させるものである。本実施形態において、アーム部は、連結された一連の部材であるアジャスタ2、キャリア23及びガイドポール66から構成される。
駆動部は、アーム部を略水平方向に駆動するものであり、本実施形態では、モータ62、ボールネジ63、ガイドシャフト64及びシャフトホルダ65によって構成されている。
制御部68は、分離転写装置100の各種制御(アーム部の位置の制御、陽極32及び陰極41に印加する電流・電圧の制御等)を行う制御盤である。制御部68は、ユーザからの入力を受けるためのボタン、スイッチや、動作状態をユーザに通知するためのランプ、表示部等を備えていてもよい。
次に、分離転写装置100におけるサンプルの電気泳動及び転写の流れについて、図1を参照して説明する。図1に示すように、サンプルの電気泳動及び転写時において、転写膜1は、アジャスタ2によって、第一開口50aに当接する位置に配置された状態で保持される。このとき、陽極バッファ槽30の底部に設けられたガイド33・34によって、転写膜1は、転写膜1の裏面(分離部50とは反対側)から支持されている。
本発明の態様1に係る転写膜保持器具(アジャスタ2)は、電気泳動によって検体を分離し、分離した該検体を排出部(第一開口50a)から排出し、転写膜1を該排出部に当接させて移動させることによって分離した該検体を該転写膜に転写する分離転写装置100において該転写膜1を保持する転写膜保持器具であって、該転写膜1の移動方向における少なくとも一方の端部を固定する固定部(クランプ20・21)を備え、該固定部は、該排出部とは反対側から該転写膜に当接する弾性体20a・20bと、該転写膜を該弾性体に押し付ける押し付け部材20b・21bとを備えている。
1a・1b 端部
2 アジャスタ(転写膜保持器具)
20 クランプ(固定部、第二の固定部、アーム部)
21 クランプ(固定部、第一の固定部、アーム部)
20a・21a 弾性体
20b・21b 押し付け部材
A 当接部
B 軸
C 取っ手部
20c・21c ストッパ(係止部材)
20d・21d 滑り止め部材
21e ロック(係止部材)
22 クランプフレーム(アーム部、連結部)
23 キャリア(アーム部、側壁の上端を回り込む部位、第二部位)
30 陽極バッファ槽(第一緩衝液槽)
31 テーブル
32 陽極(第1電極)
33・34 ガイド(支持部材)
40 陰極バッファ槽(第二緩衝液槽)
41 陰極(第2電極)
42 ロック
50 分離部
50a 第一開口(排出部)
50b 第二開口
51・53 絶縁板
52 分離ゲル(分離媒体)
62 モータ(駆動部)
63 ボールネジ(駆動部)
64 ガイドシャフト(駆動部)
65 シャフトホルダ(駆動部)
66 ガイドポール(アーム部、第一部位)
68 制御部
100 分離転写装置
Claims (11)
- 電気泳動によって検体を分離し、分離した該検体を排出部から排出し、転写膜を該排出部に当接させて移動させることによって分離した該検体を該転写膜に転写する分離転写装置において該転写膜を保持する転写膜保持器具であって、
該転写膜の移動方向における少なくとも一方の端部を固定する固定部を備え、
該固定部は、
該排出部とは反対側から該転写膜に当接する弾性体と、
該転写膜を該弾性体に押し付ける押し付け部材と
を備えていることを特徴とする転写膜保持器具。 - 上記押し付け部材は、上記移動方向と直交する、上記転写膜から上記弾性体とは反対側に離間した軸を中心に回動するようになっており、当該回動に伴って上記転写膜に当接して上記転写膜を上記弾性体に押し付ける当接部を備えていることを特徴とする請求項1に記載の転写膜保持器具。
- 上記押し付け部材は、上記回動操作のための取っ手部をさらに備えていることを特徴とする請求項2に記載の転写膜保持器具。
- 上記押し付け部材は、上記軸を曲部とするL字状の形状を有していることを特徴とする請求項3に記載の転写膜保持器具。
- 上記転写膜の移動方向における一方の端部を固定する第一の固定部を備え、
第一の固定部の押し付け部材が、上記転写膜の移動方向における他方の端部から該一方の端部に向かう方向に上記転写膜を引き摺るように回動することによって上記転写膜を上記弾性体に押し付けるようになっていることを特徴とする請求項2~4の何れか一項に記載の転写膜保持器具。 - 第一の固定部は、第一の固定部の押し付け部材の回動の範囲を規定する係止部材を備えていることを特徴とする請求項5に記載の転写膜保持器具。
- 上記他方の端部を固定する第二の固定部を備え、
第二の固定部の押し付け部材が、上記他方の端部から上記一方の端部に向かう方向に上記転写膜を引き摺るように回動することによって上記転写膜を上記弾性体に押し付けるようになっていることを特徴とする請求項5又は6に記載の転写膜保持器具。 - 第二の固定部は、第二の固定部の押し付け部材の回動の範囲を規定する係止部材を備えていることを特徴とする請求項7に記載の転写膜保持器具。
- 上記当接部に、滑り止め部材が設けられていることを特徴とする請求項2~8の何れか一項に記載の転写膜保持器具。
- 上記滑り止め部材は、上記当接部に埋め込まれていることを特徴とする請求項9に記載の転写膜保持器具。
- 電気泳動によって検体を分離し、分離した該検体を排出部から排出し、転写膜を該排出部に当接させて移動させることによって分離した該検体を該転写膜に転写する分離転写装置であって、
請求項1~10の何れか一項に記載の転写膜保持器具を備えていることを特徴とする分離転写装置。
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ES15883369T ES2856548T3 (es) | 2015-02-24 | 2015-12-07 | Dispositivo de separación y transferencia con soporte de membrana de transferencia |
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SG11201610796RA SG11201610796RA (en) | 2015-02-24 | 2015-12-07 | Transfer membrane retaining jig and separation-transfer device |
US15/303,548 US10191010B2 (en) | 2015-02-24 | 2015-12-07 | Transfer membrane retaining jig and separation-transfer device |
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EP3264075A1 (en) | 2018-01-03 |
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CN106461605B (zh) | 2018-09-11 |
KR101873143B1 (ko) | 2018-06-29 |
ES2856548T3 (es) | 2021-09-27 |
CN106461605A (zh) | 2017-02-22 |
SG11201610796RA (en) | 2017-01-27 |
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