JP2016095249A - Medium cutout tool, medium cutout device, and medium cutout method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medium cutout tool, a medium cutout device, and a medium cutout method which achieve satisfactory collection of an electrophoresed medium.SOLUTION: A medium cutout tool (2) includes a sidewall (3) which surrounds an opening (4), which forms a hollow space (5), and which is pressed against a medium containing an electrophoresed sample so as to cut out a portion facing the opening (4) in the medium. The sidewall (3) includes a first tapered part (3a) slanting toward the inside of the hollow space (5) to an end part on the side of being pressed against the medium.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a medium cutting device, a medium cutting device, and a medium cutting method for cutting out a medium containing an electrophoresed sample.

  In recent years, proteome analysis has been actively performed as a method for exhaustively analyzing proteins that directly support biological activities. Proteome is the whole protein produced in a specific cell, organ and organ, and proteome analysis is a large-scale study on the structure and function of protein. Two-dimensional electrophoresis, which is a technique for separating proteins, has high resolution and can detect thousands of proteins at a time. Therefore, it is often used as a method for separating biological samples such as proteins.

  Two-dimensional electrophoresis is a technique for two-dimensionally separating proteins by two-stage electrophoresis based on the physical properties of the protein. For example, after isolating the protein in the first dimension based on the isoelectric point by isoelectric focusing (IEF), it is further converted to molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Based on this, the proteins are separated in the second dimension. In isoelectric focusing, a voltage is applied to the gel, and the proteins are separated by the difference in isoelectric points of the proteins. Further, in SDS-PAGE, electrophoresis is performed in a state where a complex of sodium dodecyl sulfate (SDS), which is an anionic surfactant, and a protein is formed, and the protein is separated based on a difference in molecular weight or the like.

  The gel for the first dimension electrophoresis in the two-dimensional electrophoresis has an elongated strip shape with a width of 3 mm and a length of about 7 to 20 cm. This strip has a three-layer structure, and a dry polyacrylamide gel having a thickness of 100 μm or less is chemically adsorbed on a plastic film, and a protective film is formed to protect the gel. Yes. The protective film is adsorbed by the tackiness of the polyacrylamide gel, and the polyacrylamide gel is not applied to the end of the protective film.

  By two-dimensional electrophoresis, proteins are detected as spots in a flat gel. The type of protein in the detected spot can be identified by peptide mass fingerprinting (PMF). In the peptide mass fingerprint method, each protein spot on the gel is cut out, the cut gel is replaced with a trypsin treatment buffer, and then the protein in the gel is digested into peptides with a protein digestion enzyme such as trypsin. And mass spectrometry is performed with respect to the peptide eluted from the gel, and the protein before digestion is identified. For example, Patent Document 1 discloses a method of cutting out one spot of a gel containing a protein separated by two-dimensional electrophoresis and digesting the protein in the spot gel into a peptide.

  Here, in the conventional technique, a gel plug recovery device for cutting out a protein spot developed (separated) into a flat gel in a circular shape presses a cylindrical tube against the flat gel, After cutting into a circular shape, the cut gel is sucked and collected. Moreover, when discharging the cut gel, the gel is discharged by a pressure for discharging a liquid such as water or air. For example, Patent Document 2 includes a cylindrical cutout outer tube and a cutout inner tube having an outer diameter smaller than that of the cutout outer tube and having the same shape, and gas passes through the tip of the cutout inner tube. There is disclosed a spot picker device in which ventilating portions in which holes are formed at regular intervals are provided, and a ventilation pipe is provided between the outer cut-out pipe and the cut-out inner pipe for flowing clean air. In the spot picker device, after the gel was cut by lowering the cut outer tube and the cut inner tube, the inside of the cut inner tube was slightly negative pressure and cut from the fixed base by introducing clean air from the ventilation section. The gel is lifted and collected.

JP2007-215412 (released on August 30, 2007) JP 2005-77242 A (published March 24, 2005)

  As disclosed in Patent Document 2, in the method of cutting out a spot detected by electrophoresis using a tubular tube, the gel is clogged in the tubular tube during collection of the gel, or the side wall of the tube. There is a problem that the gel cannot be recovered satisfactorily due to, for example, the gel being chipped due to friction and the like.

  Accordingly, the present invention has been made in view of the above-described problems, and an object thereof is to provide a gel cutting device, a gel cutting device, and a gel cutting method that can satisfactorily recover the electrophoretic medium. There is to do.

  In order to solve the above problems, a medium cutting device according to one aspect of the present invention is a hollow member having an opening, a side wall that surrounds the opening and forms a hollow space, and is electrophoresed. A side wall that is pressed against the medium containing the sample and cuts out a portion of the medium that faces the opening, and the side wall faces the opening at the end on the opening side and is inside the hollow space. Inclined in the direction.

  According to said structure, in the medium cutting device which concerns on 1 aspect of this invention, the medium of the site | part facing an opening part is pressed by pressing the front-end | tip part (namely, opening part) of a medium cutting tool with respect to a gel. It will be cut out and taken into the hollow space. Since the tip of the medium cutting device has a tapered shape that is inclined toward the inside of the hollow space, the tip of the medium cutting device may become clogged when the cut medium is taken into the hollow space. It can be prevented from being damaged due to friction with the side wall or the like. As described above, according to the medium cutting device according to one aspect of the present invention, the electrophoresed medium can be recovered well.

  The medium cutting device according to one aspect of the present invention may be configured to include a plurality of portions each constituting a part of the side wall, and to change the relative positions of the plurality of portions. For example, the opening is circular, and a first portion having a sidewall adjacent to a portion of the circumference of the opening and a second portion having a sidewall adjacent to the remainder of the circumference of the opening. The structure provided may be sufficient.

  According to the above configuration, the medium cutting device is configured by a plurality of portions (for example, the first portion and the second portion) each constituting a part of the side wall, and the relative positions of the plurality of portions are changed. By doing so, the medium at the site where the end of the medium cutting device is pressed can be surely cut, and the medium at the desired site can be cut out.

  In the medium cutting device according to one embodiment of the present invention, after cutting out a medium at a desired site, the medium cutting device holding the cut medium is immersed in the solution in the container, and the cut medium is taken out. By changing the relative position of the plurality of parts in a state where the medium cutting device is immersed in the solution in the container at least to the end of the medium cutting device, the inside of the hollow space is changed according to the change in the size of the hollow space. The water level of the solution changes. The gel plug in the hollow space can be scraped off by the vertical flow of the solution that occurs as the water level of the solution changes.

  In the medium cutting device according to an aspect of the present invention, the side wall has an inclined portion that is inclined toward the opening side toward the inner side of the hollow space at a position different from the end portion on the opening side. Furthermore, the structure which it has may be sufficient.

  According to the above configuration, when the cut out medium is taken out, the plurality of the above pieces are immersed in the solution in the container up to the inclined portion formed at a position different from the end of the medium cutting device. By changing the relative positions of the portions, the flow of the solution in the container becomes intense due to the tapered shape formed at the other position. Thereby, since the change in the water level of the solution in the hollow space also becomes severe, the medium in the hollow space can be taken out more effectively.

  In the medium cutting device according to one aspect of the present invention, at least one of a protrusion and a depression for preventing the medium from adhering is formed on the inner surface of the side wall at the end on the opening side. There may be.

  According to said structure, the contact area of the medium cutting device and the cut-out medium can be made small, and it can prevent that the cut-out medium adheres to a medium cutting device.

  In order to solve the above-described problem, a medium cutting device according to one aspect of the present invention includes any one of the above-described medium cutting devices, a drive unit that moves the medium cutting device relative to the medium, Is provided. In particular, it is preferable that the medium cutting device includes a plurality of portions each constituting a part of the side wall, and the drive unit is capable of changing a relative position between the plurality of portions. Further, the medium cutting device controls the driving unit to press the medium cutting tool against the medium, and changes the relative positions of the plurality of parts in a state where the medium cutting device is pressed against the medium. The structure which further provided the part may be sufficient. Further, the side wall of the medium cutting device further includes an inclined portion that is inclined inward of the hollow space toward the opening side at a position different from the end portion on the opening side. The medium cutting device controls the drive unit to convey the medium cutting device into a specific container in which the solution is placed, and sets the relative positions of the plurality of parts in the specific container. The structure further provided with the 2nd control part to change may be sufficient.

  According to the above configuration, it is possible to automatically perform from the cutting out of the medium to the taking out.

  In order to solve the above-described problem, a medium cutting method according to one aspect of the present invention is a medium cutting method using any one of the above-described medium cutting devices, wherein the side wall is pressed against the medium, and the medium cutting method is performed. A cutting step of cutting out a portion of the medium facing the opening, and a holding step of holding the cut-out medium in the hollow space after the cutting step.

  According to said method, there exists an effect similar to the medium cutting device which concerns on 1 aspect of this invention.

  According to one embodiment of the present invention, an electrophoretic medium can be recovered favorably.

It is a side view of the medium cutting device which concerns on one Embodiment of this invention. It is a block diagram of the medium cutting-out apparatus which concerns on one Embodiment of this invention. It is a figure which shows the gel containing the sample electrophoresed. (A) And (b) in a figure is a top view and sectional drawing when changing the relative position of two parts which comprise the medium cutting device which concerns on one Embodiment of this invention. (A)-(d) in a figure is a figure which shows each process of the medium cutting-out method by the medium cutting-out apparatus which concerns on one Embodiment of this invention. (A) And (b) in a figure is a figure which shows each process of the medium removal method by the medium cutting-out apparatus which concerns on one Embodiment of this invention. (A) And (b) in a figure is a figure which shows each process of the medium cutting-out method by the medium cutting-out apparatus which concerns on one Embodiment of this invention. (A) And (b) in a figure is a figure which shows each process of the medium cutting-out method by the medium cutting-out apparatus which concerns on one Embodiment of this invention. (A)-(c) in a figure is a shape example of the inner surface of the 1st taper-shaped part of the medium cutting device which concerns on one Embodiment of this invention. It is an actual drawing of the medium cutting-out apparatus which concerns on one Embodiment of this invention. It is a real photograph of the gel after cut out, and the cut out gel plug.

  Hereinafter, a medium cutting device and a medium cutting device according to an embodiment of the present invention will be described in detail with reference to FIGS. 1 and 2.

In addition, for example, when collecting the gel after two-dimensional electrophoresis with a conventional tubular medium cutting tool,
(1) The gel is likely to clog the tube of the medium cutting device. (2) Since the gel is sucked and recovered by air pressure or the like, the recovered gel is chipped due to friction with the side wall of the tube of the medium cutting device. Easy (3) Since the thinner the tube of the medium cutting device, the easier it is to clog and break, there is a problem that it is difficult to recover a gel block having a diameter of less than 1 mm, and the gel cannot be recovered well. According to an embodiment of the present invention, at least one of the above problems can be solved.

(Configuration of the medium cutting device 1)
FIG. 1 is a side view of a medium cutting device according to the present embodiment, and FIG. 2 is a block diagram of the medium cutting device according to the present embodiment. Hereinafter, a mode in which the medium cutting device according to this embodiment is incorporated in the medium cutting device according to this embodiment will be described. However, the medium cutting device according to this embodiment is used alone, for example, manually operated. May be.

  The medium cutout device according to the present embodiment is a device for cutting out a specific region including spots or bands in a medium containing an electrophoresed sample. For example, FIG. 3 shows a gel after electrophoresis as a medium containing an electrophoresed sample. Each spot 8 of the gel 6 contains proteins separated by electrophoresis. The medium cutting device according to the present embodiment is used to cut out a specific area including each spot 8 of the gel 6. Hereinafter, the medium cutting device according to the present invention will be described by taking as an example a mode of cutting out a specific region of a gel containing an electrophoresed sample using the medium cutting device. However, the present invention is not necessarily limited to this. For example, when a separated sample is transferred from a gel to a transfer film, the medium cutting device according to the present invention is used to cut out a specific area of the transfer film. You can also.

  As illustrated in FIG. 2, the medium cutting device 1 includes a medium cutting device 2 and a control device 10 (first control unit, second control unit). The control device 10 moves the medium cutting device 2 with respect to the gel 6 placed on the stage 7. The control device 10 can move the medium cutting device 2 in the XYZ directions (in the horizontal plane and in the vertical direction). Note that the movement of the medium cutting device 2 by the control device 10 means a relative movement, and both the mode for moving the medium cutting device 2 and the mode for moving the stage 7 on which the gel 6 is placed are implemented. Included in the form.

  As shown in FIG. 1, the medium cutting device 2 is a hollow member having an opening 4, a side wall 3 that surrounds the opening 4 and forms a hollow space 5, and includes a gel containing an electrophoresed sample. 6 and has a side wall 3 for cutting out a portion of the gel 6 facing the opening 4. The side wall 3 has a first taper-shaped portion 3 a that is inclined toward the inside of the hollow space 5 toward the opening 4 at the end on the side pressed against the gel 6 (that is, the end on the opening 4 side). Have.

  In FIG. 1, the medium cutting device 2 is configured by a plurality of portions (a first medium cutting device 2 </ b> A and a second medium cutting device 2 </ b> B) each constituting a part of the side wall 3. The control device 10 changes the relative positions of the plurality of parts (the first medium cutting device 2A and the second medium cutting device 2B). In the present embodiment, the medium cutting device 2 is composed of two parts. However, even if a medium cutting device composed of three or more parts is used, the same effects as those described in this specification are obtained. Can be obtained.

(Configuration of media cutting device)
The configuration of the medium cutting device 2 will be described in more detail with reference to FIG. 4A and 4B are a top view and a cross-sectional view when the relative positions of the two portions constituting the medium cutting device 2 are changed.

  As shown to (a) in FIG. 4, the opening part 4 of the medium cutting device 2 is circular, and the medium cutting device 2 has a side wall 3 adjacent to a part of the circumference of the opening 4. An instrument 2A (first part) and a second medium cutting instrument 2B (second part) having a side wall 3 adjacent to the remaining part of the circumference of the opening 4 are provided. The size of the opening 4 can be set to a diameter of 100 μm to 500 μm, for example. Note that the opening 4 is not circular, but can have the same effects as those described in the present specification even in a square or rectangular shape.

  As shown in FIG. 4A, at least one of the first medium cutting device 2A and the second medium cutting device 2B is moved in a direction opposite to the other, and the side wall 3 of the first medium cutting device 2A. And the end of the side wall 3 of the second medium cutting device 2B are combined to form a circular opening by the side wall 3 of the first medium cutting device 2A and the side wall 3 of the second medium cutting device 2B. 4 and the hollow space 5 are formed. Hereinafter, a state in which the end of the side wall 3 of the first medium cutting device 2A and the end of the side wall 3 of the second medium cutting device 2B are combined is referred to as a “closed state”.

  In this closed state, the relative positions of the first medium cutting device 2A and the second medium cutting device 2B are changed, and the distance between the two side walls 3 is changed as shown in FIG. 4B. Hereinafter, a state in which the relative positions of the first medium cutting device 2A and the second medium cutting device 2B are changed to increase the distance between the two side walls 3 is referred to as an “open state”.

  As described above, in the first medium cutting device 2A and the second medium cutting device 2B, each side wall 3 is inclined toward the inner side of the hollow space 5 toward the opening 4 at the end on the opening 4 side. The first tapered portion 3a is provided. Further, in the first medium cutting device 2A and the second medium cutting device 2B, each side wall 3 is located at a position different from the end portion on the opening 4 side, toward the opening 4 side and inward of the hollow space 5. It has the 2nd taper-shaped part 3b (inclination part) which inclines toward. The functions of the first tapered portion 3a and the second tapered portion 3b will be described in detail later.

  In the first medium cutting device 2A and the second medium cutting device 2B, each side wall 3 is formed with a grip portion 3c at an end opposite to the end on the opening 4 side. Each gripping part 3c fixes each of the first medium cutting device 2A and the second medium cutting device 2B to a support member (not shown) that supports the first medium cutting device 2A and the second medium cutting device 2B. belongs to. As shown in FIG. 4, each grip portion 3c is formed with a plurality of screw holes 11 for fastening screws when the first medium cutting device 2A and the second medium cutting device 2B are fixed to the support member. It may be.

  The medium cutting device 2 can be moved by connecting the support member to which the medium cutting device 2 is fixed to the moving device (drive unit) and the control device 10 controlling the moving device. For example, a gripper can be used as the support member, and a stepping motor can be used as the moving device.

(Media cutout method)
A medium cutting method by the medium cutting device 1 will be described with reference to FIG. (A)-(d) in FIG. 5 is a figure which shows each process of the medium cutting-out method by the medium cutting-out apparatus 1. FIG.

  First, a specific region including a spot or band of the gel 6 is specified. The method for specifying the specific region is not particularly limited. For example, the stained spot or band may be visually specified by the user by performing visible staining or fluorescent staining of the gel 6 subjected to electrophoresis. Alternatively, the spot or band may be automatically identified using software that detects the stained spot or band.

  After the specific area is specified, the medium cutting device 2 of the medium cutting device 1 is moved immediately above the specific area. The moving method of the medium cutting device 2 is not particularly limited. For example, the user inputs an instruction to move the medium cutting device 2 from the outside to the medium cutting device 1, and based on the movement instruction, the control device 10 causes the medium cutting device 2 (or The stage 7) may be moved. Alternatively, when the specific area is automatically specified using software, the control device 10 may move the medium cutting device 2 (or stage 7) based on the information on the specific area specified by the software.

  When the control device 10 moves the medium cutting device 2 directly above the specific region and makes the opening 4 of the medium cutting device 2 face the specific region, the control device 10 is as shown in FIG. The medium cutting device 2 is moved downward. At this time, the medium cutting device 2 is in a closed state. The control device 10 moves the medium cutting device 2 downward, and presses the end of the first tapered portion 3a (that is, the end on the opening 4 side) against the gel 6.

  Even if the end portion of the first tapered portion 3a is pressed against the gel 6, the gel 6 is not cut immediately because it is an elastic body, but is stretched and cut by the pressed pressure. Therefore, the gel 6 that has been cut has a restoring force after being cut and contracts, and is cut more than the area formed by the end portion of the first tapered portion 3a (that is, the size of the opening 4). The gel 6 can be made small.

  Since the gel 6 is an elastic body, if the end of the first tapered portion 3a is pressed once against the gel 6, the gel 6 is not sufficiently cut, and the gel 6 may be left uncut. Therefore, the control device 10 moves at least one of the first medium cutting device 2A and the second medium cutting device 2B in a direction opposite to the other from the state of FIG. 5A in which the medium cutting device 2 is pressed against the medium. Then, as shown in FIG. The distance for moving at least one of the first medium cutting device 2A and the second medium cutting device 2B in a direction opposite to the other can be set to about 200 μm to 500 μm, for example. The control device 10 alternately repeats the closed state of FIG. 5 (a) and the open state of FIG. 5 (b), so that the gel at the site where the end of the first tapered portion 3a is pressed 6 is cut reliably and the gel plug 9 is cut out. In this way, the gel plug 9 at a portion (that is, a specific region) facing the opening 4 can be cut out.

  In the medium cutting device 1, the end of the first taper-shaped portion 3 a of the medium cutting device 2 (that is, the end on the opening 4 side) is pressed against the gel 6, so that the portion of the portion facing the opening 4 is pressed. The gel plug 9 is cut out and taken into the hollow space 5. Since the distal end portion of the medium cutting device 2 has a taper shape, when the cut gel plug 9 is taken into the hollow space 5, the distal end portion of the medium cutting device 2 is clogged, or friction with the side wall 3 occurs. It can be prevented from being damaged due to chipping or the like. Thus, according to the medium cutting-out device 1, the gel 6 that has been electrophoresed can be recovered satisfactorily. Further, in the conventional tubular cutting device, the thinner the tube, the easier it is to clog and break the gel plug cut into the tube, so it is difficult to recover the gel plug having a diameter of 1 mm or more. However, in the medium cutting device 1 according to this embodiment, a gel plug having a diameter of less than 1 mm can be recovered without any problem.

  Note that the solution 12 is thinly stretched on the gel 6. In this state, the control device 10 alternately repeats the closed state shown in FIG. 5A and the open state shown in FIG. 5B, whereby the medium cutting device 2 is shown in FIG. The solution 12 enters the hollow space 5, and the cut gel plug 9 is taken into the hollow space 5 along the flow of the solution 12. Thus, in the medium cutting device 2, the gel plug 9 can be easily collected.

  Furthermore, since the tip of the medium cutting device 2 has a tapered shape, the gel plug 9 taken into the hollow space 5 is held by the first tapered portion 3a. Therefore, as shown in FIG. 5D, the control device 10 moves the medium cutting device 2 while holding the gel plug 9 in a specific area between the first medium cutting device 2A and the second medium cutting device 2B. Then, the gel plug 9 can be transported to the collection container without dropping by gravity.

(Media removal method)
A method of taking out the medium by the medium cutting device 1 will be described with reference to FIG. (A) and (b) in FIG. 6 is a diagram showing each step of the medium cutout method by the medium cutout device 1.

  The control device 10 transports the medium cut and held by the medium cutting device 2 into the specific container 17, and then the medium cutting device 2 is placed in the container by the control device 10 as shown in FIG. Immerse in the solution 13 in 17. In FIG. 6A, the tip of the medium cutting device 2 is put in the container 17 until at least the first tapered portion 3 a is immersed in the solution 13. At this time, the medium cutting device 2 is in a closed state.

  In the container 17, the control device 10 moves at least one of the first medium cutting device 2A and the second medium cutting device 2B from the state shown in FIG. ) Open as shown. The control device 10 alternately repeats the closed state shown in FIG. 6A and the open state shown in FIG. 6B, so that the solution in the hollow space 5 changes according to the change in the size of the hollow space 5. 13 water levels change. The gel plug 9 in the hollow space 5 is scraped off by the vertical flow of the solution 13 that occurs with the change in the water level of the solution 13. Thus, the gel plug 9 cut out and collected can be reliably taken out into the container 17 using the flow of the solution 13.

  In the present embodiment, medium cutting and medium removal are realized by the medium cutting tool 2 constituted by the two medium cutting tools 2A and 2B. However, three or more plural pieces each constituting a part of the side wall. The same effect can be obtained even if a medium cutting device having the above portion is used.

  Further, the gel 6 chips adhering to the inner surface of the side wall 3 of the medium cutting device 2 due to the vertical flow of the solution 13 caused by the change in the water level of the solution 13 in the hollow space 5 of the medium cutting device 2 are also generated. Removed. In this way, the control device 10 cleans the inside of the hollow space 5 of the medium cutting device 2 by alternately repeating the closed state of FIG. 6 (a) and the open state of FIG. 6 (b). Can also be fulfilled.

  A preferred form when the medium is taken out by the medium cutting device 1 will be described with reference to FIG. 7A and 7B are diagrams showing each step of the medium cutting out method by the medium cutting out device 1. FIG.

  As shown in (a) of FIG. 7, when the medium cutting device 2 is immersed in the solution 13 in the container 17 by the control device 10, the medium cutting device 2 is kept until the second tapered portion 3 b is immersed in the solution 13. The tip is preferably placed in the container 17. The control device 10 moves at least one of the first medium cutting device 2A and the second medium cutting device 2B from the state of FIG. 7A in a direction opposite to the other, as shown in FIG. 7B. In the open state, the size of the hollow space 5 is increased. When the control device 10 alternately repeats the closed state of FIG. 7A and the open state of FIG. 7B, the flow of the solution 13 in the container 17 is intensely caused by the second tapered portion 3b. Become. Thereby, since the change of the water level of the solution 13 in the hollow space 5 also becomes intense, the gel plug 9 in the hollow space 5 can be taken out more effectively.

  With reference to FIG. 8, a description will be given of a further preferred mode at the time of taking out the medium by the medium cutting device 1. FIGS. 8A and 8B are diagrams showing each step of the medium cutting-out method by the medium cutting-out apparatus 1.

  As shown in FIG. 8A, the control device 10 puts the tip of the medium cutting device 2 into the container 17 until the second tapered portion 3b is immersed in the solution 13, and closes the medium cutting device 2 in this state. It is preferable to move the medium cutting device 2 up and down in the container 17 as shown in FIG. 8B while alternately repeating the state and the open state. Moving up and down means changing the vertical distance between the medium cutting device 2 and the container 17 by moving the medium cutting device 2 relative to the container 17. By moving the medium cutting device 2 up and down in the container 17 while alternately repeating the medium cutting device 2 between the closed state and the open state, the flow of the solution 13 in the container 17 becomes more intense. Thereby, since the change of the water level of the solution 13 in the hollow space 5 also becomes intense, the gel plug 9 in the hollow space 5 can be taken out more effectively.

(Example of shape of first tapered portion 3a)
Here, FIG. 9 shows a shape example of the inner surface of the first taper-shaped portion 3a of each of the first medium cutting device 2A and the second medium cutting device 2B (that is, the inner surface at the end on the opening 4 side). Since the gel 6 has adhesiveness, when the gel plug 9 cut out between the first medium cutting device 2A and the second medium cutting device 2B is held, the gel plug 9 is formed on the inner surface of the first tapered portion 3a. There is a possibility of adhering. Therefore, it is preferable that the inner surfaces of the first tapered portion 3a of each of the first medium cutting device 2A and the second medium cutting device 2B have a shape for preventing the gel plug 9 from adhering.

  For example, as shown to (a) of FIG. 9, the protrusion 14 for preventing that the gel plug 9 adheres may be formed in the inner surface of the 1st taper-shaped part 3a. There is no particular limitation on the size and shape of the protrusion, and as shown in FIG. 9B, a protrusion 15 smaller than the protrusion 14 may be formed on the inner surface of the first tapered portion 3a. Alternatively, as shown in FIG. 9C, a recess 16 for preventing the gel plug 9 from adhering to the inner surface of the first tapered portion 3a may be formed. There is no particular limitation on the size and shape of the recess.

  By forming such protrusions or depressions on the inner surface of the first tapered portion 3a, the contact area between the inner surface of the first tapered portion 3a and the gel plug 9 can be reduced. Can be prevented from adhering to the inner surface. Note that the shape for preventing the gel plug 9 from adhering is not limited to the above-described protrusion or depression. Further, both the above-described protrusions and depressions may be formed on the inner surface of the first taper-shaped part 3a, and at least one of the above-described protrusions and depressions is formed on the inner surface of the first taper-shaped part 3a. Is possible.

(Example)
Below, the suitable example at the time of utilizing the medium cutting-out apparatus 1 which concerns on this embodiment is described with reference to FIG. 10 and FIG. FIG. 10 is a real photograph of the medium cutting device 1, and FIG. 11 is a real photograph of the cut gel and cut gel plug.

  The medium cutting apparatus 1 according to the present embodiment is used for cutting out a gel subjected to electrophoresis such as sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional electrophoresis. In SDS-PAGE or two-dimensional electrophoresis, gels of various sizes having a thickness of about 1 mm and a size of up to about 24 cm × 24 cm are used. In either case, proteins are separated according to molecular weight by a molecular sieving effect using a flat acrylamide gel. The separated protein is detected as a rectangular band by SDS-PAGE and can be detected as a spot by two-dimensional electrophoresis. Below, the Example at the time of utilizing the medium cutting-out apparatus 1 which concerns on this embodiment in two-dimensional electrophoresis is described.

  First, a mouse liver soluble protein is prepared as a sample, and Bench Mark (registered trademark) (manufactured by Life Technology) is prepared as a molecular weight marker (a plurality of peptides having a known molecular weight).

  Isoelectric focusing is performed by placing an IPG gel strip in an electrophoresis chamber and applying a voltage to the first electrode and the second electrode that are in contact with the IPG gel strip. Specifically, isoelectric focusing is performed by mixing 8M urea, 2M thiourea, 4% CHAPS (3-[(3-Cholamidopropylo) dimethylamylopropanolate), 20 mM dithiothreitol, 0.5% Amphorite in a sample mixture. Then, after the IPG gel strip is swollen with the swelling liquid mixed with the sample, a voltage is applied. The voltage is gradually increased by maintaining 200V for 5 minutes, increasing from 200V to 1000V in 5 minutes, maintaining 1000V for 5 minutes, increasing from 1000V to 6000V in 10 minutes, and maintaining 6000V for 5 minutes. To raise the protein to the isoelectric point. By isoelectric focusing, the protein of the sample migrates in the longitudinal direction of the IPG gel strip to a position corresponding to each isoelectric point (pI).

  Next, the IPG gel strip is introduced into the second-dimensional electrophoresis apparatus, and the second-dimensional electrophoresis is performed. Specifically, the IPG gel strip was deposited in an aqueous solution containing 50 mM DTT, 500 mM Tris-HCl (pH 6.6), 4% SDS, 12.5% Glycerol, 0.005% BPB for 5 minutes, Install the IPG gel strip in the sample loading section. Further, a molecular weight marker is applied to the side of the IPG gel strip, a voltage is applied at 20 mA between the cathode and the anode, and electrophoresis is performed for about 30 minutes.

  When the second-dimensional electrophoresis is completed and stained with Coomassie-Brilliant-Blue, the separated proteins are visualized as spots. As shown in FIG. 10, the spot detected by the two-dimensional electrophoresis is targeted, and the spot is cut out from the gel using the medium cutting device 1 according to the present embodiment. By using the medium cutting device 1 according to the present embodiment, a gel plug (for example, a diameter of about 0.7 mm) as shown by A in FIG. 11 is cut out. B shown in FIG. 11 is a trace of the gel plug cut out.

The gel plug of the cut out spot is transferred to a container such as a microtube or a microplate, and the protein in the gel is digested. Specifically, 50 μL of 100% acetonitrile (CH ₃ CN) and 50 μL of 50 mM ammonium hydrogen carbonate (NH ₄ HCO ₃ ) are added, and the mixture is allowed to stand at room temperature for 15 minutes for destaining. Next, 100 μL of 100% CH ₃ CN was added and allowed to stand at room temperature for 10 minutes, 10 mM DTT, 50 mM NH ₄ HCO ₃ was added, and the mixture was incubated at 60 ° C. for 10 minutes, and then allowed to stand for 20 minutes. Reduce. Next, 30 μL of 50 mM iodoacetamide and 50 mM NH ₄ HCO ₃ are added and left to stand at room temperature for 15 minutes for alkylation. Next, 40 μL of 50 mM NH ₄ HCO ₃ was added and allowed to stand at room temperature for 15 minutes. Further, 100 μL of 100% CH ₃ CN was added and allowed to stand at room temperature for 30 minutes, and then the liquid in the well was discharged. And put under nitrogen for 10 minutes. Next, 25 μL of endopeptidase (eg, trypsin digestion solution) is added and incubated overnight at 30 ° C., and the extracted peptide is used as a sample for the mass spectrometer.

  The above sample is analyzed using a mass spectrometer, and the protein is identified by the obtained peptide data. Specific methods include MASCOT, X! This is done using software that collates with a database, such as Tandem, OMASASA or Android.

  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. Furthermore, a new technical feature can be formed by combining the technical means disclosed in each embodiment.

  The present invention is suitably used in the field of separating a biological sample such as protein by electrophoresis, cutting out a gel containing the separated biological sample, and identifying or analyzing the biological sample in the gel with a mass spectrometer. Can do.

DESCRIPTION OF SYMBOLS 1 Medium cutting device 2 Medium cutting device 2A 1st medium cutting device 2B 2nd medium cutting device 3 Side wall 3a 1st taper-shaped part (end part by the side of an opening part)
3b 2nd taper shape part (inclination part)
4 Opening 5 Hollow space 6 Gel (medium)
7 stage 8 spot 9 gel plug 10 control device (first control unit, second control unit)
11 Screw hole 12 Solution 13 Solution 14 Protrusion 15 Protrusion 16 Depression 17 Container (specific container)

Claims (10)

A hollow member having an opening,
A side wall that surrounds the opening and forms a hollow space, the side wall being pressed against a medium containing the electrophoresed sample, and a side wall for cutting out a portion of the medium facing the opening;
The medium cutting device according to claim 1, wherein the side wall is inclined toward the inside of the hollow space toward the opening at the end on the opening side. Each comprising a plurality of portions constituting part of the side wall;
The medium cutting device according to claim 1, wherein the relative positions of the plurality of portions can be changed. The opening is circular,
A first portion having a side wall adjacent to a portion of the circumference of the opening, and a second portion having a side wall adjacent to the remaining portion of the circumference of the opening. 2. The medium cutting device according to 2.   The side wall further includes an inclined portion that is inclined toward the inside of the hollow space toward the opening side at a position different from the end portion on the opening side. The medium cutting device according to 2 or 3.   5. At least one of a protrusion and a depression for preventing the medium from adhering is formed on the inner surface of the side wall at the end on the opening side. The medium cutting device according to Item. The medium cutting device according to any one of claims 1 to 5,
And a drive unit that moves the medium cutting device relative to the medium. The medium cutting device comprises a plurality of parts each constituting a part of the side wall,
The medium cutting device according to claim 6, wherein the driving unit is capable of changing a relative position between the plurality of portions.   A first control unit configured to control the driving unit to press the medium cutting device against the medium and change a relative position between the plurality of parts in a state of pressing the medium cutting tool against the medium; The medium cutting device according to claim 7, wherein The side wall of the medium cutting device further includes an inclined portion that is inclined inward of the hollow space toward the opening side at a position different from the end portion on the opening side,
The medium cutting device controls the driving unit to transport the medium cutting device into a specific container in which a solution is placed, and changes the relative positions of the plurality of parts in the specific container. The medium cutting device according to claim 7, further comprising a second control unit for causing the medium to be cut out. A medium cutting method using the medium cutting device according to any one of claims 1 to 5,
A cutting step of pressing the side wall against the medium to cut out a portion of the medium facing the opening;
A medium cutting method comprising: a holding step of holding the cut medium in the hollow space after the cutting step.