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WO2015064736A1 - Milieu d'électrophorèse unidimensionnelle pour électrophorèse bidimensionnelle, support sur lequel est maintenu le milieu, dispositif d'électrophorèse bidimensionnelle utilisant le milieu, procédé d'électrophorèse bidimensionnelle utilisant le milieu et gabarit - Google Patents

Milieu d'électrophorèse unidimensionnelle pour électrophorèse bidimensionnelle, support sur lequel est maintenu le milieu, dispositif d'électrophorèse bidimensionnelle utilisant le milieu, procédé d'électrophorèse bidimensionnelle utilisant le milieu et gabarit Download PDF

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Publication number
WO2015064736A1
WO2015064736A1 PCT/JP2014/079040 JP2014079040W WO2015064736A1 WO 2015064736 A1 WO2015064736 A1 WO 2015064736A1 JP 2014079040 W JP2014079040 W JP 2014079040W WO 2015064736 A1 WO2015064736 A1 WO 2015064736A1
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Prior art keywords
dimensional electrophoresis
medium
electrophoresis
dimensional
gel
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English (en)
Japanese (ja)
Inventor
英樹 木下
祥之 石田
公彦 矢部
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Sharp Corp
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Sharp Corp
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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
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • the present invention relates to a first-dimensional electrophoretic medium used for two-dimensional electrophoresis, and more specifically, a first-dimensional electrophoretic gel capable of realizing high analysis accuracy and high reproducibility, and a medium holding the medium
  • the present invention relates to an attached holder, a two-dimensional electrophoresis apparatus using the medium, and a two-dimensional electrophoresis method using the medium.
  • proteome analysis A technique for comprehensively analyzing proteins in a living body is called proteome analysis.
  • proteome analysis two-dimensional electrophoresis is often used because it can detect protein differences with images.
  • Two-dimensional electrophoresis is known as a method for separating proteins based on the physical properties of isoelectric point and molecular weight. Since all proteins have unique charges and molecular weights, they can be separated into various proteins by separating the protein mixture present in the living body by molecular weight or charge. In particular, separation by charge is important because even if the protein is the same type of protein, there are protein species with different charges due to post-translational modification, and the molecular weights of these proteins are almost the same.
  • the above-described two-dimensional electrophoresis method is a primary method in which isoelectric focusing is performed by immersing a sample as a sample in an immobilized pH gradient (IPG) gel and then applying a voltage to both ends of the IPG gel.
  • IPG immobilized pH gradient
  • SDS-PAGE method Polyacrylamide gel electrophoresis
  • SDS sodium dodecyl sulfate
  • a sample by migrating a standard substance (sometimes called a marker) to be compared with the sample together with the sample.
  • a standard substance sometimes called a marker
  • an isoelectric point marker migrates with a sample in the case of the above-described isoelectric focusing
  • a molecular weight marker migrates with a sample in the case of SDS-PAGE.
  • these standard substances are applied to the gel together with the sample at the start of electrophoresis. That is, an isoelectric point marker is applied to the first dimension electrophoresis gel at the start of the first dimension electrophoresis, and a molecular weight marker is applied to the second dimension electrophoresis gel at the start of the second dimension electrophoresis.
  • isoelectric markers are mixed with a sample and soaked in an IPG gel.
  • molecular weight markers are generally applied manually to a portion where the IPG gel does not exist after soaking in SDS. It is.
  • the present invention has been made in view of the above-described problems, and its purpose is to simplify the work of the electrophoretic practitioner and to perform two-dimensional electrophoresis with high reproducibility.
  • electrophoresis medium holding body with medium holding the medium, two-dimensional electrophoresis apparatus using the medium, two-dimensional electrophoresis method using the medium, and jig for producing the medium Is to provide.
  • first-dimensional electrophoresis of the two-dimensional electrophoresis is referred to as first-dimensional electrophoresis
  • second-dimensional electrophoresis is referred to as second-dimensional electrophoresis. That is, two-dimensional electrophoresis is constituted by the first-dimensional electrophoresis and the subsequent second-dimensional electrophoresis.
  • a first-dimensional electrophoresis medium for two-dimensional electrophoresis is a first-dimensional electrophoresis medium used for first-dimensional electrophoresis of two-dimensional electrophoresis. It is characterized in that it contains no electrophoresis sample and contains a standard substance to be compared with the electrophoresis sample.
  • a two-dimensional electrophoresis apparatus includes a pair of electrodes for first-dimensional electrophoresis, a cathode and an anode for second-dimensional electrophoresis, A two-dimensional electrophoresis apparatus that contains a molecular weight standard as a reference material for comparison of electrophoresis samples in one area and a medium containing the electrophoresis sample in the other area.
  • the pair of electrodes for the first-dimensional electrophoresis used as an electrophoresis medium is configured to apply an electric field to a region that does not contain the molecular weight standard in the first-dimensional electrophoresis medium.
  • the cathode and anode for electrophoresis are characterized in that an electric field is applied to the entire region of the first-dimensional electrophoresis medium.
  • FIG. 1 is a perspective view of an isoelectric focusing gel that is an embodiment of a first-dimensional electrophoretic medium for two-dimensional electrophoresis according to the present invention. It is a figure of the holding body with a gel provided with the isoelectric focusing gel shown in FIG. 2, (a) is a side view, (b) is a side view by the side of an isoelectric focusing gel. It is the figure which illustrated the process in the middle of producing the isoelectric focusing gel shown in FIG. It is the figure which illustrated the process in the middle of producing the isoelectric focusing gel shown in FIG.
  • FIG. 3 is a top view of the isoelectric focusing gel shown in FIG. 2.
  • FIG. 2 shows an isoelectric focusing instrument performed using the isoelectric focusing gel shown in FIG. 2, wherein (a) is a top view of the isoelectric focusing instrument, and (b) is an isoelectric focusing instrument. It is sectional drawing which showed the state which set the isoelectric focusing gel. It is a figure which shows one Embodiment of the two-dimensional electrophoresis apparatus which concerns on this invention. It is sectional drawing of the two-dimensional electrophoresis apparatus shown in FIG. FIG.
  • FIG. 6 is a perspective view of an isoelectric focusing gel that is another embodiment of the first-dimensional electrophoresis medium of the two-dimensional electrophoresis according to the present invention.
  • transducing a sample and a molecular weight marker simultaneously into the isoelectric focusing gel shown in FIG. 11 is shown, (a) is a top view, (b) is the cutting line A shown to (a).
  • FIG. It is a figure which shows a mode that a sample and a molecular weight marker are simultaneously introduce
  • FIG. 12 is a cross-sectional view of an isoelectric focusing instrument for performing isoelectric focusing using the isoelectric focusing gel shown in FIG. 11 in which a sample and a molecular weight marker are introduced. It is a figure which shows other embodiment of the two-dimensional electrophoresis apparatus which concerns on this invention. It is a figure which shows the electrophoresis result of an Example. It is a figure which shows the electrophoresis result of an Example.
  • Embodiment 1 Hereinafter, an embodiment of the present invention will be described in detail with reference to FIGS.
  • the present invention relates to a first-dimensional electrophoresis medium for two-dimensional electrophoresis, a holding body with a medium for holding the medium, a two-dimensional electrophoresis apparatus using the medium, and a two-dimensional electrophoresis method using the medium. Therefore, in the first embodiment, two-dimensional electrophoresis in which isoelectric focusing is performed as the first-dimensional electrophoresis and SDS-PAGE is performed as second-dimensional electrophoresis is given.
  • an isoelectric point marker is applied to the isoelectric focusing gel at the start of isoelectric focusing, and the SDS-PAGE gel is started at the start of SDS-PAGE.
  • Apply a molecular weight marker For this reason, the SDS-PAGE operator inevitably performs the molecular weight marker application operation, which is not only complicated, but depending on the skill of the operator, the molecular weight marker cannot be applied satisfactorily. However, there is a risk that the analysis accuracy may be lowered or reproducibility may not be achieved.
  • a standard substance (molecular weight marker) used in the second-dimensional electrophoresis is used as the first-dimensional electrophoresis gel (isoelectric focusing gel). )
  • the molecular weight marker may be a protein mixture used as a reference for molecular weight.
  • FIG. 1 shows a state in which the isoelectric focusing gel 1 (isoelectric focusing medium) is in contact with the end of the SDS-PAGE gel 424 on the cathode side. This state is shown in FIG. The time point at which SDS-PAGE electrophoresis as electrophoresis is started is shown. Characteristically, a marker introduction region 2 is formed at one end of the isoelectric focusing gel 1 so that an electric field is not applied during the first-dimensional electrophoresis and an electric field is applied during the second-dimensional electrophoresis.
  • the molecular weight marker (molecular weight standard) used in the second-dimensional electrophoresis is introduced into the marker introduction region 2 before the first-dimensional electrophoresis.
  • This molecular weight marker does not move until the second-dimensional electrophoresis, but remains in the marker introduction region 2, while when the second-dimensional electrophoresis starts, it is separated by the first-dimensional electrophoresis as indicated by an arrow in FIG. It moves from the isoelectric focusing gel 1 to the SDS-PAGE gel 424 together with the electrophoresis sample (white circle in FIG. 1) (the molecular weight marker is a black circle in FIG. 1) and migrates in the SDS-PAGE gel 424. It has a configuration.
  • the timing of introducing the molecular weight marker into the isoelectric focusing gel is such that the sample as an electrophoresis sample (hereinafter sometimes simply referred to as a sample) is introduced before being introduced into the isoelectric focusing gel.
  • the sample is introduced into the isoelectric focusing gel simultaneously with the sample. Therefore, in the first embodiment, the former aspect will be described, and the latter aspect will be described in the second embodiment described later.
  • FIG. 2 is a perspective view of the isoelectric focusing gel of the first embodiment.
  • the isoelectric focusing gel 1 of Embodiment 1 has a band shape, and a marker introduction region 2 into which a molecular weight marker is introduced is formed at one end region thereof. Further, an isoelectric focusing region 3 into which a sample is introduced is formed next to the marker introducing region 2 along the longitudinal direction of the strip-shaped isoelectric focusing gel 1.
  • Embodiment 1 has already had a molecular weight marker in the marker introduction region 2 at a stage before the isoelectric focusing sample is introduced into the isoelectric focusing gel 1.
  • the introduced isoelectric focusing gel is provided. That is, Embodiment 1 provides an isoelectric focusing gel 1 in which the isoelectric focusing region 3 does not include a sample and is composed only of an electrophoresis gel, and a molecular weight marker is introduced only into the marker introduction region 2. It is.
  • the practitioner of the two-dimensional electrophoresis only needs to obtain the isoelectric focusing gel 1 of Embodiment 1 and introduce the sample into the isoelectric focusing region 3, and the molecular weight marker at the start of the second dimensional electrophoresis. There is an advantage that the implementer does not have to introduce it.
  • the isoelectric focusing gel 1 of Embodiment 1 is preferably, for example, an immobilized pH gradient (IPG) gel.
  • the IPG gel is, for example, an acrylamide / bisacrylamide mixed solution that forms a gel skeleton (for example, a mixing ratio: 37.5 / 1), several acrylamide buffers that define the pH in the gel skeleton, and a polymerization initiator.
  • the strip-shaped isoelectric focusing gel 1 of Embodiment 1 has a long length that is the same as or shorter than the length of the cathode side edge of the SDS-PAGE gel described later. Thereby, the contact structure shown in FIG. 1 is realizable.
  • the isoelectric focusing gel 1 of Embodiment 1 may be in a dry state or in a swollen state, but the standard substance or sample is present rather than existing in the swollen gel. Since it is more stable in the gel in the dry state, it is preferably in the dry state for distribution and storage.
  • the present invention also includes a holding body with a gel in which a belt-like first-dimensional electrophoresis gel is adhered to the holding surface of the holding portion. Then, the holding body 40 with a gel of this Embodiment 1 is shown in FIG. In FIG. 3, a gel-attached holding body 40 is provided in which the belt-shaped isoelectric focusing gel 1 shown in FIG. 2 is bonded to the holding surface of the holding unit 4.
  • A) in FIG. 3 is a side view of the holding body 40 with gel
  • (b) in FIG. 3 is a side view of the holding body 40 with gel on the side of the isoelectric focusing gel 1.
  • the holding part 4 may be made of an insulating material having a chip-like shape, for example, or may be a film.
  • the strip-shaped isoelectric focusing gel 1 adhered to the holding surface of the holding unit 4 may be in a swollen state or in a dry state.
  • the marker introduction method in Embodiment 1 includes a gel preparation step for preparing an isoelectric focusing gel sheet before introduction of a molecular weight marker, and a molecular weight on a part of the isoelectric focusing gel sheet prepared by the gel preparation step.
  • Gel preparation process In the gel preparation step for preparing an isoelectric focusing gel sheet before introduction of the molecular weight marker, first, an acrylamide / bisacrylamide mixed solution (mixing ratio: 37.5 / 1) and four acrylamide derivatives (acid dissociation constant: 3.6). 6.2, 7.0, 8.5), ammonium persulfate (APS), tetramethylethylenediamine (TEMED; N, N, N ′, N′-tetramethylethylenediamine), glycerol and pure water, Prepare an acidic monomer solution and a basic monomer solution adjusted at a desired mixing ratio (for example, PG Righetti: Immobilized pH gradients: theory and methodology, Elsevier, Amste rdam, 1990).
  • PG Righetti Immobilized pH gradients: theory and methodology, Elsevier, Amste rdam, 1990.
  • the prepared acidic-side monomer solution and basic-side monomer solution are mixed using a mixer, and the obtained mixed solution is filled into an IPG gel preparation device and gelled.
  • a sheet-like gel adhesion layer (for example, a gel bond layer) is previously put on an IPG gel preparation instrument. A film) is placed, and the mixed solution is filled therein.
  • the length of the obtained IPG gel can be arbitrarily designed from 50 mm to 240 mm, for example.
  • a protective film is attached to the surface of the IPG gel opposite to the gel adhesive layer.
  • a chip adhesive layer is provided on the gel adhesive layer.
  • an adhesive may be applied on the gel adhesive layer, or a double-sided tape may be applied.
  • a roller or the like it is preferable to use a roller or the like carefully so that air bubbles and dust do not enter.
  • a sheet-like isoelectric focusing gel having a protective film provided on one side is formed.
  • a generally square sheet-shaped isoelectric focusing gel is formed.
  • This square sheet-shaped isoelectric focusing gel has a shape in which a plurality of strip-shaped isoelectric focusing gels finally produced are arranged. In this way, by producing a plurality of strip-shaped isoelectric focusing gels in a sheet at a time, the processing performed in the marker introduction step described later can be performed efficiently, and handling is also easy. preferable.
  • the shape of the sheet-like isoelectric focusing gel is not limited to this.
  • a step of introducing a molecular weight marker into a part of the sheet-like isoelectric focusing gel prepared in the gel preparation step is performed.
  • the molecular weight marker is placed in the immersed region by immersing one side of the square sheet-like isoelectric focusing gel in the marker-containing liquid containing the molecular weight marker and the vicinity thereof.
  • the one side of the sheet-like isoelectric focusing gel immersed in the molecular weight marker solution is a side along the direction orthogonal to the pH gradient direction of the IPG gel prepared in the gel preparation step.
  • FIG. 4 is a perspective view of a marker introduction jig 100 (jig) used in the marker introduction process.
  • the marker introduction jig 100 shown in FIG. 4 has three rows of groove portions provided in parallel, and the groove portion located in the center row of them stores a marker liquid storage portion 102 (standard substance) in which a molecular weight marker solution is stored.
  • the remaining two grooves located on the front and rear sides of the marker liquid storage part 102 are marker liquid escape parts 101 that receive the marker liquid overflowing from the marker liquid storage part 102.
  • one side of the sheet-like isoelectric focusing gel and the vicinity thereof are immersed in the molecular weight marker solution in the marker solution reservoir 102.
  • the sheet-like isoelectric focusing gel is supported by a support member (not shown), and the lower end region of the sheet-like isoelectric focusing gel is immersed in the molecular weight marker solution as shown in FIG.
  • a molecular weight marker solution can be infiltrated into only the lower end region, and a sheet-like isoelectric focusing gel in which a marker introduction region is formed in a part of the sheet-like isoelectric focusing gel is prepared easily and reliably. (FIG. 5).
  • the sheet-like isoelectric focusing gel immersed in the molecular weight marker solution in the marker introduction step may be in a swollen state or in a dry state.
  • a drying step is provided after the marker introduction step and before the subsequent cutting step.
  • the molecular weight marker is stably held in the lower end region of the sheet-like isoelectric focusing gel, and can be easily cut during the subsequent cutting step.
  • a strip-shaped isoelectric focusing gel is cut out from the sheet-shaped isoelectric focusing gel in which the molecular weight marker is introduced on one side and the vicinity thereof.
  • one side of the sheet-like isoelectric focusing gel into which the molecular weight marker is introduced is a side along a direction orthogonal to the pH gradient direction of the IPG gel prepared in the gel preparation step. Therefore, a band-shaped isoelectric focusing gel in which a molecular weight marker is introduced into one end region of the band-shaped isoelectric focusing gel is obtained by cutting along the pH gradient direction so as to divide the one side into a plurality of parts. It can be produced (FIG. 6).
  • an isoelectric focusing gel into which the molecular weight marker shown in FIG. 2 has been introduced is completed.
  • the marker introduction region 2 realized in one end region of the strip-shaped isoelectric focusing gel 1 applies an electric field to the strip-shaped isoelectric focusing gel 1 during the isoelectric focusing.
  • the pair of electrodes 301 are positioned away from the electrodes. Therefore, while the voltage is applied to the pair of electrodes 301, that is, while the sample is being subjected to isoelectric focusing, the molecular weight marker introduced into the marker introduction region 2 is within the isoelectric focusing gel 1. None move.
  • the holding body with gel 40 shown in FIG. 3 is produced by adhering the isoelectric focusing gel cut into a band shape by the above-described cutting process to the holding part 4. Can do.
  • the holding surface of the holding unit 4 is adhered to the upper surface of the sheet-like isoelectric focusing gel into which the molecular weight marker is introduced, and simultaneously with the cutting, the holding with gel shown in FIG.
  • the body 40 should be completed. This is because if the band-like isoelectric focusing gel is handled alone, there is a high risk of bending or tearing. It is preferable to handle not only the holding part 4 but also the band-like isoelectric focusing gel in a state of being bonded to some member.
  • Embodiment 1 (1-5) Sample Introduction Method to Isoelectric Focusing Electrophoresis Gel of Embodiment 1
  • the strip-like isoelectric focusing electricity shown in FIG. 2 is used by using the sample introduction jig 200 shown in FIG. A sample is introduced into the isoelectric focusing region 3 of the electrophoresis gel 1.
  • this Embodiment 1 although the method to introduce a sample into the strip
  • the sample introduction jig 200 includes a housing 203 that is provided with a groove 202 for holding the sample liquid 201 at the bottom and opens upward.
  • the gel-attached holding body 40 is inserted from the opening side of the housing 203 toward the bottom, and the isoelectric focusing region 3 of the band-shaped isoelectric focusing gel 1 is held in the groove 202.
  • the sample solution 201 comes into contact with the sample solution 201, the sample solution 201 is absorbed and the sample is introduced into the isoelectric focusing region 3.
  • the isoelectric focusing gel 1 is preferably dried to such an extent that the sample liquid 201 can be absorbed.
  • the sample introduction jig 200 also serves as an isoelectric focusing device to be described later, even if the isoelectric focusing gel 1 is in a swollen state, the sample liquid 201 and the isoelectric focusing region 3 is in contact, the electric field is applied to the isoelectric focusing region 3 and the sample moves into the isoelectric focusing region 3 at the same time.
  • the electrophoresis gel 1 does not have to be in a dry state.
  • the sample introduction is performed for the purpose of moving the sample in the sample liquid 201 into the isoelectric focusing region 3.
  • the sample may be introduced by applying an electric field to the isoelectric focusing region 3 by a pair of electrodes provided on the jig 200.
  • the isoelectric focusing gel 1 is also in contact with the sample liquid 201. Need not be dry.
  • sample introduction method is not limited to this method.
  • a sample liquid is placed on the surface of the isoelectric focusing area 3 using a pipetter or the like, and the sample is introduced into the isoelectric focusing area 3. Also good.
  • the sample introduction jig 200 can also be configured as an isoelectric focusing instrument to be described later. That is, sample introduction and isoelectric focusing can be combined with one jig (instrument). Thus, when sample introduction and sample isoelectric focusing are performed in the sample introduction jig 200, rapid sample analysis becomes possible.
  • FIG. 8 shows isoelectric focusing that performs isoelectric focusing (first-dimensional electrophoresis step) using the isoelectric focusing gel 1 of the first embodiment.
  • FIG. 9 is a diagram showing the instrument, in which (a) in FIG. 8 is a top view of the isoelectric focusing instrument, and (b) in FIG. 8 is the isoelectric point along the cutting line AA ′ shown in (a). It is sectional drawing of an electrophoresis instrument.
  • the isoelectric focusing gel 1 is not shown in FIG. 8A, and the isoelectric focusing gel 1 is shown in FIG.
  • a pair of electrodes 301 is arranged on the bottom surface of a recess provided in the tank body 302.
  • the concave portion of the tank body 302 has a strip-like shape along the bottom surface.
  • Each of the pair of electrodes 301 is formed by arranging a linear conductor along the short direction of the recess as shown in FIG.
  • the pair of electrodes 301 can be made of platinum, for example.
  • the electrodes 301 are separated from each other by a predetermined length in the longitudinal direction of the concave portion of the tank body 302.
  • the predetermined length that is, the separation distance is equal to or slightly smaller than the length along the longitudinal direction (that is, the pH gradient direction) of the isoelectric focusing region 3 of the band-shaped isoelectric focusing gel 1. It is configured to be short.
  • the band-shaped isoelectric focusing gel 1 has a length in the longitudinal direction (that is, pH gradient direction) of the isoelectric focusing region 3 equal to the distance between the pair of electrodes 301 or the pair of electrodes 301. It is configured to be slightly longer than the separation distance.
  • the gap between the bottom surface of the recess of the tank body 302 and the strip-shaped isoelectric focusing gel 1. are filled with a buffer solution 303 for isoelectric focusing.
  • the dried strip-shaped isoelectric focusing gel 1 absorbs the buffer solution 303 and swells.
  • the buffer solution 303 is not filled in the bottom surface of the recess of the tank body 302.
  • the buffer solution 303 is also in contact with the marker introduction region 2. This is because when the strip-shaped isoelectric focusing gel 1 installed in the isoelectric focusing instrument 300 is in a dry state, the buffer solution 303 is brought into contact with only the isoelectric focusing region 3. This is because it is assumed that the surface of the isoelectric focusing gel 1 becomes uneven when it only swells and good isoelectric focusing cannot be performed. By bringing the buffer solution 303 into contact with the marker introduction region 2 and causing it to swell together with the isoelectric focusing region 3, good isoelectric focusing can be performed.
  • the marker introduction region 2 may be brought into contact with the buffer solution 303 or not.
  • the isoelectric focusing gel 1 of the state with which the holding body 40 with a gel shown in FIG. Although it is set to 300, it is not limited to this method.
  • the sample introduction jig 200 when the sample introduction jig 200 is provided with a pair of electrodes, the sample introduction jig 200 can also have the function of the isoelectric focusing instrument 300.
  • the isoelectric focusing instrument 300 is described as an instrument for first-dimensional electrophoresis out of two-dimensional electrophoresis.
  • the isoelectric focusing instrument 300 is provided as a dedicated electrophoresis apparatus for isoelectric focusing. May be. That is, an electrophoresis device dedicated to isoelectric focusing using the isoelectric focusing gel 1 of Embodiment 1 is also included in the scope of the present invention.
  • FIG. 9 shows a main configuration of the automated two-dimensional electrophoresis apparatus 400 according to the first embodiment.
  • FIG. 9 is a perspective view showing a main configuration of the automated two-dimensional electrophoresis apparatus 400.
  • B in FIG. 9 shows a configuration in which the isoelectric focusing gel 1 described above is coupled to the support arm 431 via the holding unit 4.
  • C in FIG. 9 is a cross-sectional view showing a configuration of a second-dimensional electrophoresis unit 420 used in the automated two-dimensional electrophoresis apparatus 400.
  • the automated two-dimensional electrophoresis apparatus 400 includes a one-dimensional electrophoresis unit 410 including the isoelectric focusing instrument 300 described above on a fixing means 401 serving as a pedestal, and an SDS-PAGE (second dimension A second-dimensional electrophoresis unit 420 for performing the electrophoresis step), the support arm 431, and a driving unit 404 that moves the fixing unit 401 and / or the support arm 431 to change the relative positions of the two. ing.
  • the sample is first separated in the first direction (Y direction in FIG. 9) by the isoelectric focusing instrument 300 of the one-dimensional electrophoresis unit 410, and the sample is equilibrated. Later, the separation is performed in the second direction (X direction in FIG. 9) by the second-dimensional electrophoresis unit 420.
  • This is realized by the driving means 404 holding the support arm 431 so as to be movable in the X-axis direction and the Z-axis direction.
  • the one-dimensional electrophoresis unit 410 is provided with a plurality of tanks, one of which is configured as an isoelectric focusing instrument 300 including the pair of electrodes 301 described above.
  • the one-dimensional electrophoresis unit 410 will be further described with reference to FIG.
  • FIG. 10 is a cross-sectional view of the automated two-dimensional electrophoresis apparatus 400.
  • the one-dimensional electrophoresis unit 410 has a configuration in which a plurality of tanks 411 and 412 are provided in a single insulator.
  • the first reagent tank 411 is for storing reagents necessary for the process until the first dimension separation
  • the second reagent tank 412 is after the first dimension separation and before the second dimension separation. It is for storing necessary reagents.
  • the first reagent tank 411 includes a gel arrangement tank 411a, a sample tank 411b, a swelling tank 411c, and a first separation tank 411d.
  • the first separation tank 411 d is configured as an isoelectric focusing instrument 300, and a voltage is applied to the isoelectric focusing gel 1 by the first voltage applying unit 405 through a pair of electrodes 301.
  • the sample tank 411b has a sample introduction part and is configured as a sample introduction jig 200 (FIG. 7).
  • the second reagent tank 412 includes a first equilibration tank 412a, a staining tank 412b, a washing tank 412c, and a second equilibration tank 412d.
  • the first equilibration tank 412a is preferably provided to store a buffer solution for replacing the buffer solution used for the first direction separation and increasing the efficiency of staining performed after the first direction separation.
  • the washing tank 412c is preferably provided for storing a buffer solution for washing excess fluorescent dye attached in the staining tank 412b in which the fluorescent dye is stored.
  • the second equilibration tank 412d stores a reagent preferable for performing the second direction separation.
  • a reagent for reducing the protein of the isoelectric focusing gel 1 and a reagent for converting the protein into SDS are stored.
  • a buffer solution, a surfactant, an enzyme, an interacting substance, or the like may be stored depending on the second direction separation method.
  • the driving means 404 first drives the support arm 431 to the desired X position of the gel placement tank 411a, and then lowers the support arm 431 to the desired Z position. Then, adsorption
  • the adsorption control to the support arm 431 can be automatically controlled by using, for example, an electromagnetic valve.
  • the driving unit 404 moves the gel-attached holding body 40 adsorbed on the support arm 431 in the direction of the arrow 402 in FIG. 10, whereby the isoelectric focusing gel 1 is provided in the one-dimensional electrophoresis unit 410. Each of the tanks is subjected to a desired process and subsequently conveyed to the second-dimensional electrophoresis unit 420.
  • the step of introducing the sample into the isoelectric focusing region 3 of the isoelectric focusing gel 1, the step of swelling the isoelectric focusing gel 1, and the isoelectric focusing Applying a voltage to the gel 1 to separate the sample in the first direction in the isoelectric focusing region 3, and separating the sample in the isoelectric focusing gel 1 (in the isoelectric focusing region 3)
  • a step of staining and a step of equilibrating to the environment in the second-dimensional electrophoresis unit 420 are performed.
  • the one-dimensional electrophoresis unit 410 by adding the sample to the isoelectric focusing gel 1 and swelling the isoelectric focusing gel 1 separately, the total time of sample introduction and swelling speed is obtained. This can be shortened compared to the conventional case.
  • the second-dimensional electrophoresis unit 420 includes an SDS-PAGE gel 424, which is included in the isoelectric focusing gel 1 conveyed from the first-dimensional electrophoresis unit 410 in the SDS-PAGE gel 424.
  • the separated sample is further separated (SDS-PAGE) in a second direction different from the first direction.
  • the second-dimensional electrophoretic unit 420 includes a first buffer tank 428a and a first buffer tank 428a provided in the lower insulating unit through the upper insulating plate 422 and an insulating unit 420a in which the lower insulating plate 421 and the upper insulating plate 422 are overlapped. 2 buffer tank 428b.
  • the lower insulating plate 421 is provided with a gel storage portion 424 ′ for covering and storing the SDS-PAGE gel 424 between the lower insulating plate 421 and the upper insulating plate 422.
  • the SDS-PAGE gel 424 stored in the gel storage unit 424 ′ is covered with an insulating part 420 a composed of a lower insulating plate 421 and an upper insulating plate 422, and the first opening 425 and the second opening 426 have the insulating part 420 a Can contact the outside.
  • the first opening 425 and the second opening 426 face the first buffer tank 428a and the second buffer tank 428b provided in the second-dimensional electrophoresis unit 420, respectively.
  • the first buffer solution tank 428a and the second buffer solution tank 428b include the SDS-PAGE gel 424 and the first opening 425 stored in the gel storage unit 424 ′.
  • the first buffer solution and the second buffer solution that are in contact with each other through the second opening 426 are filled.
  • the first buffer tank 428a and the second buffer tank 428b are provided with a first electrode 429a and a second electrode 429b, and SDS is applied by the second voltage applying means 406 via the first electrode 429a and the second electrode 429b.
  • the width of the opening through the upper insulating plate 422 in the second buffer solution tank 428b is the groove of the corresponding lower insulating plate 421. Wider than width. Due to this difference, as shown in FIG. 10, the isoelectric focusing gel 1 of the gel-attached holding body 40 inserted from the second opening 426 and the SDS-PAGE gel 424 can be brought into close contact with each other. The sample in the isoelectric focusing gel 1 after the original electrophoresis can be successfully separated by SDS-PAGE.
  • the insulating portion 420a covering the SDS-PAGE gel 424 brings the isoelectric focusing gel 1 and the SDS-PAGE gel 424 into close contact with each other.
  • Such a portion may be the second opening 426 or a further opening 426 ′ may be provided between the first opening 425 and the second opening 426.
  • the SDS-PAGE gel 424 protrudes from the second opening 426, in order to increase the degree of adhesion. It is more preferable that the protruding SDS-PAGE gel 424 has no irregularities.
  • an adhesive member for bringing the isoelectric focusing gel 1 and the SDS-PAGE gel 424 into close contact with the second opening 426 (Not shown) may be provided.
  • Preferred adhesive members include, but are not limited to, agarose, low viscosity (about 1-3%) gels such as acrylamide, and high viscosity liquids such as glycerin, polyethylene glycol, and hydroxypropyl cellulose.
  • the fixing to the fixing means 401 is detachable on the assumption that the one-dimensional electrophoresis unit 410, the second-dimensional electrophoresis unit 420, and the gel-attached holding body 40 may be exchanged for each sample.
  • a pinch fixing mechanism, a magnetic force fixing mechanism, and an electrostatic adsorption mechanism can be cited in addition to the vacuum adsorption mechanism, but not limited thereto.
  • a cooling means 409 (for example, a heat radiating means) shown in FIG. 10 may be provided immediately below the fixing means 401.
  • the temperatures of the one-dimensional electrophoresis unit 410 and the second-dimensional electrophoresis unit 420 during electrophoresis can be kept constant.
  • sample described above may be a substance to be separated or analyzed by electrophoresis and transcription.
  • a biological material such as an individual organism, body fluid, cell line, tissue culture or tissue fragment.
  • the collected preparation can be suitably used.
  • Particularly preferred are polypeptides or polynucleotides.
  • Two-dimensional electrophoresis With respect to the two-dimensional electrophoresis apparatus shown in FIG. 9, the separation in the first direction (Y direction in the figure) and the separation in the second direction (X direction in the figure) are specified.
  • the parameters may be the same, but are preferably different in order to improve the separation performance.
  • Parameters that define separation in these two directions include protein isoelectric point, molecular weight, surface charge per unit size (zone electrophoresis), distribution coefficient to micelles (micellar electrokinetic chromatography), stationary phase-transfer Examples include the partition coefficient to phases (electrochromatography) and affinity constants for interacting substances (affinity binding electrophoresis).
  • separation in the first direction is based on the isoelectric point.
  • the separation in the second direction is performed based on the molecular weight.
  • (1-10) Marker As the molecular weight marker to be introduced into the isoelectric focusing gel in Embodiment 1, a conventionally known molecular weight marker can be used, and the molecular weight range of the marker, the number of detection bands, etc. are suitable depending on the sample. A marker can be selected. It is also possible to use a plurality of molecular weight markers.
  • a known substance may be introduced into an isoelectric focusing gel as described above as a marker (which may be fluorescently labeled).
  • the molecular weight marker may be a protein mixture used as a reference for molecular weight. This means that when performing Western blotting using two-dimensional electrophoresis, the same sample as that used for two-dimensional electrophoresis Western blotting is added to the position where the molecular weight marker of the isoelectric focusing gel is inserted. It is assumed. As a result, the pattern when the sample is separated only by SDS-PAGE can be compared with the pattern when added by two-dimensional electrophoresis.
  • the mode in which the marker for the second-dimensional electrophoresis is introduced into the first-dimensional electrophoresis gel has been described.
  • the present invention is not limited to this, and a marker for the first-dimensional electrophoresis (for example, an isoelectric point marker) is previously introduced into the first-dimensional electrophoresis gel, and if necessary, The gel can be dried and stored, and when the first-dimensional electrophoresis of the sample becomes necessary, it is also possible to introduce the sample into the gel with the isoelectric marker already introduced and perform the first-dimensional electrophoresis. is there.
  • an object of the present invention is to eliminate the trouble of an electrophoresis practitioner introducing a marker into a gel and to solve a problem that is a concern when the practitioner introduces a marker. Therefore, a gel in which a marker used in the first-dimensional electrophoresis is previously introduced into the first-dimensional electrophoresis gel is also a means for realizing this purpose.
  • the marker used in the first-dimensional electrophoresis is introduced in advance into the first-dimensional electrophoresis gel, the marker needs to move in the gel by the first-dimensional electrophoresis.
  • the introduction position needs to be within the pair of electrodes described above.
  • Target gel into which a marker is introduced In the first embodiment, a molecular weight marker is introduced into an isoelectric focusing gel.
  • the target gel into which a marker is introduced is not limited to an isoelectric focusing gel. Any first-dimensional electrophoretic medium may be used as long as it is used for first-dimensional electrophoresis.
  • FIG. 11 is a perspective view of the isoelectric focusing gel of the second embodiment.
  • the difference from the isoelectric focusing gel 1 of the first embodiment (FIG. 2) is that the band-shaped isoelectric focusing gel 1 ′ of the second embodiment has molecular weight markers introduced at both ends.
  • the marker introduction region 2 is formed.
  • a region sandwiched between these marker introduction regions 2 is an isoelectric focusing region 3.
  • the isoelectric focusing gel 1 ′ of the second embodiment can also be handled in the form of the gel-attached holding body 40 of FIG. 3 as in the first embodiment.
  • FIG. 12 is a diagram showing a simultaneous introduction jig 500 (jig) for introducing a molecular weight marker and a sample used in the second embodiment, and (a) in FIG. 12 is a top view of the simultaneous introduction jig 500.
  • FIG. 12B is a cross-sectional view of the simultaneous introduction jig 500 taken along a cutting line AA ′ in FIG.
  • FIG. 13 is a diagram illustrating a method for introducing a molecular weight marker and a sample into an isoelectric focusing gel using the simultaneous introduction jig 500, and corresponds to (b) in FIG.
  • the simultaneous introduction jig 500 is configured as a casing 501 in which elongated grooves are formed.
  • the elongated grooves (medium holding portions) have a length along the length of the strip-shaped isoelectric focusing gel. It is formed almost equally.
  • a plurality of depressions are formed at the bottom of the groove, and a marker liquid depression 502 (a substance holding part for introducing a standard substance) that stores molecular weight marker liquid at both ends of the elongated groove, a marker liquid depression 502, and a marker
  • a sample solution recess 503 epitrophoretic sample introduction substance holding unit for storing the sample solution is formed at approximately the center of the elongated groove sandwiched between the solution recesses 502.
  • FIG. 13 shows a state in which the liquid is stored in each of these depressions.
  • the sample solution 533 is stored in the sample solution recess 503
  • the molecular weight marker solution 552 is stored in the marker solution recess 502, and the marker solution recess 502 and the sample solution recess 503 are stored.
  • Buffer solution 554 is stored in a recess provided at the boundary.
  • the isoelectric focusing gel is made to oppose the bottom part of the housing
  • the isoelectric focusing gel at the time of introduction is a band-shaped isoelectric point before the introduction of the sample having a band shape.
  • the electrophoresis gel can be produced by a method including the gel preparation step and the cutting step described in the item (1-3) of the first embodiment.
  • the isoelectric focusing device 300 for performing isoelectric focusing using the isoelectric focusing gel 1 ′ is the isoelectric focusing device 300 of the first embodiment. As shown in the figure, a pair of electrodes is provided, and the distance between the electrodes is equal to or slightly shorter than the length along the longitudinal direction of the isoelectric focusing region 3 (that is, the pH gradient direction). In addition, any isoelectric focusing instrument may be used as long as each of these electrodes is in contact with the isoelectric focusing region 3 of the isoelectric focusing gel 1 ′.
  • an isoelectric focusing instrument having the function of simultaneously introducing a molecular weight marker and a sample by providing the pair of electrodes 301 described above at the bottom of the simultaneous introduction jig 500 shown in FIG. 12. It can also be realized as 600. Note that the simultaneous introduction jig 500 and the isoelectric focusing instrument may be separate.
  • FIG. 15 is a perspective view of the two-dimensional electrophoresis apparatus of the second embodiment, and (a), (b), and (c) in FIG. 15 indicate the process of two-dimensional electrophoresis in this order. .
  • a two-dimensional electrophoresis apparatus 700 shown in FIG. 15 is composed of a housing 702 provided with a relatively large recess 701, and in a first direction (in a position closer to the left side of the page than the center of the recess 701).
  • a pair of electrodes 301 (for first-dimensional electrophoresis) separated from each other in the Y direction in FIG. 15 are provided.
  • a first electrode for second-dimensional electrophoresis for separating a sample in the second direction (X direction in FIG. 15) on the left side and the right side of the side surface of the concave portion 701 across the central portion of the concave portion 701. 429a and a second electrode 429b are provided.
  • a concave groove 703 is formed at the bottom of the concave portion 701 along the first direction (Y direction in FIG. 15) in the region where the pair of electrodes 301 are arranged. Has been.
  • FIG. 15 shows a configuration at the time of first-dimensional electrophoresis (isoelectric focusing), and an isoelectric focusing gel having a marker introduction region 2 and an isoelectric focusing region 3.
  • maintained 1 ' has shown the state set in the recessed part 701.
  • the SDS-PAGE gel 424 which is the second-dimensional electrophoresis gel, is then subjected to isoelectric focusing as shown in FIG. It is provided adjacent to the electrophoresis gel 1 ′. Then, after introducing a buffer solution for electrophoresis into the recess 701, a voltage is applied to the first electrode 429a and the second electrode 429b, so that the sample in the isoelectric focusing region 3 becomes a gel for SDS-PAGE. After moving to 424, the gel is separated in the second direction (X direction in FIG. 15) in the SDS-PAGE gel 424. The process up to this point is the second-dimensional electrophoresis (electrophoresis by SDS-PAGE).
  • the first-dimensional electrophoretic medium (isoelectric focusing gel 1, 1 ′) is a first-dimensional electrophoretic used for the first-dimensional electrophoresis (isoelectric focusing) of two-dimensional electrophoresis.
  • Electrophoresis medium (isoelectric focusing gel 1, 1 ′) which does not contain an electrophoresis sample (sample), but contains a standard substance (molecular weight marker) to be compared with the electrophoresis sample (sample). It is characterized by having.
  • the first-dimensional electrophoresis medium (isoelectric focusing gel 1, 1 ′) already contains the standard substance (molecular weight marker). Therefore, if it is a practitioner who performs the conventional two-dimensional electrophoresis, it is necessary to perform an operation of introducing the standard substance into the electrophoresis medium at the start of the first-dimensional electrophoresis and the second-dimensional electrophoresis. .
  • the first-dimensional electrophoretic medium isoelectric focusing gels 1, 1 ′
  • at least one of the introduction operations can be omitted, and the operation is simplified.
  • the standard substance contained in the first-dimensional electrophoresis medium is a standard substance (isoelectric point marker) used in the first-dimensional electrophoresis. If there is, the practitioner of the two-dimensional electrophoresis obtains the first-dimensional electrophoresis medium (isoelectric focusing gel 1, 1 ′), and introduces only the electrophoresis sample into the first-dimensional electrophoresis medium. The operation may be performed, and the operation for introducing the standard substance (molecular weight marker) used in the first-dimensional electrophoresis is unnecessary, and the operation of the practitioner can be simplified.
  • the standard substance contained in the first-dimensional electrophoresis medium is a standard substance (molecular weight marker) used in the second-dimensional electrophoresis
  • two The operator of the two-dimensional electrophoresis can omit the operation of introducing the standard substance at the start of the second-dimensional electrophoresis, and the operation is simplified.
  • the standard substance contained in the first-dimensional electrophoresis medium is the standard substance (molecular weight marker) used in the second-dimensional electrophoresis
  • the primary substance Primary eye electrophoresis and second-dimensional electrophoresis can be performed continuously.
  • the standard substance is a molecular weight standard (molecular weight marker) in aspect 1.
  • the first-dimensional electrophoretic medium according to aspect 3 of the present invention is in a dry state in aspect 1 or 2.
  • the first-dimensional electrophoresis medium is dry, it is possible to stably hold the standard substance (molecular weight marker).
  • the standard substance is an isoelectric point standard
  • the first-dimensional electrophoresis medium is in a dry state.
  • the first-dimensional electrophoresis medium according to aspect 5 of the present invention is the isoelectric focusing medium in the above aspects 1 to 4.
  • the first-dimensional electrophoresis medium according to Aspect 6 of the present invention has a region where an electric field is applied and a region where an electric field is not applied in the first-dimensional electrophoresis in the Aspect 2, and the region where the electric field is not applied. Contains the above molecular weight standards.
  • the contained molecular weight standard does not move in the first-dimensional electrophoresis medium during the first-dimensional electrophoresis. That is, the position of the molecular weight standard does not change even after the first-dimensional electrophoresis is finished. Therefore, by providing a region where the above electric field is not applied to the first-dimensional electrophoresis medium at a position adjacent to the end of the lane where the molecular weight standard is to be migrated in the second-dimensional electrophoresis medium, and containing the molecular weight standard therein.
  • the molecular weight standard can be moved to the second dimensional electrophoresis medium by the second dimensional electrophoresis without mixing with the electrophoresis sample separated by the first dimensional electrophoresis medium. it can.
  • the holder with a medium according to the seventh aspect of the present invention has a medium holding surface having a belt shape, and holds the first-dimensional electrophoresis medium of the first to sixth aspects along the medium holding surface. It is characterized by.
  • the first-dimensional electrophoresis medium is a thin layer or elongated and may be broken or shredded when handled alone, it may not be easy to handle, Since the first-dimensional electrophoresis medium is held on the medium holding surface of the holding body, handling becomes easy.
  • the medium-attached holding body according to aspect 8 of the present invention is the above-described aspect 7, in which the first-dimensional electrophoretic medium is at least one end portion of both end portions sandwiching the length of the medium holding surface.
  • the first-dimensional electrophoresis medium has a region where an electric field is applied during the first-dimensional electrophoresis, and is different from the region containing the standard material. Have.
  • a two-dimensional electrophoresis apparatus is a two-dimensional electrophoresis apparatus comprising a pair of electrodes for first-dimensional electrophoresis, a cathode and an anode for second-dimensional electrophoresis.
  • a medium containing a molecular weight standard as a reference substance to be compared with an electrophoresis sample in a certain area and containing the electrophoresis sample in another area is used as the first dimension electrophoresis medium.
  • the pair of electrodes for eye electrophoresis is configured to apply an electric field to a region not containing the molecular weight standard in the first dimensional electrophoresis medium, and the cathode and anode for the second dimensional electrophoresis are:
  • the present invention is characterized in that an electric field is applied to the entire region of the first-dimensional electrophoretic medium.
  • the operation of the operator since the operator who performs the two-dimensional electrophoresis does not need to introduce the molecular weight standard in the second-dimensional electrophoresis, the operation of the operator can be simplified.
  • the molecular weight standard is contained in the first-dimensional electrophoresis medium and moved from the first-dimensional electrophoresis medium to the second-dimensional electrophoresis medium during the second-dimensional electrophoresis, the first-dimensional electrophoresis medium is used.
  • the electrophoresis sample moving to the second-dimensional electrophoresis medium and the electrophoresis conditions can be made equal, and accurate analysis can be realized.
  • the first-dimensional electrophoresis medium has a band shape, and the electrode of the pair of electrodes for the first-dimensional electrophoresis is between the electrodes. The distance is shorter than the length of the strip-shaped first-dimensional electrophoresis medium and shorter than the length of the cathode-side end for the second-dimensional electrophoresis.
  • the two-dimensional electrophoresis method includes a preparatory step of impregnating an electrophoretic sample into the first-dimensional electrophoretic medium of the first to sixth aspects, and a first-dimensional electrophoretic medium following the preparatory step.
  • a first-dimensional electrophoresis step in which first-dimensional electrophoresis is performed by applying an electric field to the first-dimensional electrophoresis medium after the first-dimensional electrophoresis step is brought into contact with an end of the second-dimensional electrophoresis medium
  • a second-dimensional electrophoresis step in which a second-dimensional electrophoresis is performed by applying an electric field to the second-dimensional electrophoresis medium.
  • the electrophoresis sample in the preparation step, is placed in a region to which the electric field of the first-dimensional electrophoresis medium according to claim 6 is applied. Introducing the first-dimensional electrophoresis step, an electric field is applied to a region where the electric field is applied.
  • a jig according to the thirteenth aspect of the present invention is a jig for producing the first-dimensional electrophoretic medium, wherein the introduction substance holding unit holding the introduction substance containing the standard substance, and the standard substance A medium holding unit for holding the first-dimensional electrophoretic medium before being introduced at a position adjacent to the standard substance introducing substance holding unit is provided.
  • the jig according to the fourteenth aspect of the present invention is the jig according to the thirteenth aspect, further including an electrophoresis sample introduction substance holding unit that holds the electrophoresis sample introduction substance containing the electrophoresis sample, The holding unit holds the first-dimensional electrophoresis medium at a position adjacent to the electrophoresis sample introduction substance holding unit.
  • the standard substance and the electrophoresis sample can be introduced into the first-dimensional electrophoresis medium with one jig.
  • the jig of the thirteenth or fourteenth aspect is used to introduce an electrophoresis sample into a partial region of the first-dimensional electrophoresis medium, and A preparatory step of introducing a molecular weight standard as a standard substance to be compared with the electrophoretic sample into other regions of the electrophoretic medium, and after the preparatory step, an electric field is applied to the partial region to apply the first-dimensional electricity
  • a first-dimensional electrophoresis step of performing electrophoresis and after the first-dimensional electrophoresis step, the first-dimensional electrophoresis medium is in contact with the end of the second-dimensional electrophoresis medium on the cathode side.
  • a second-dimensional electrophoresis step of performing second-dimensional electrophoresis so that an electric field is applied to the entire region of the electrophoresis medium.
  • the operation of the operator since the operator who performs the two-dimensional electrophoresis does not need to introduce the molecular weight standard in the second-dimensional electrophoresis, the operation of the operator can be simplified.
  • the molecular weight standard is contained in the first-dimensional electrophoresis medium and moved from the first-dimensional electrophoresis medium to the second-dimensional electrophoresis medium during the second-dimensional electrophoresis, the first-dimensional electrophoresis medium is used.
  • the electrophoresis sample moving to the second-dimensional electrophoresis medium and the electrophoresis conditions can be made equal, and accurate analysis can be realized.
  • the first-dimensional electrophoresis medium for two-dimensional electrophoresis is a first-dimensional electrophoresis medium used for first-dimensional electrophoresis of two-dimensional electrophoresis, It is characterized by not containing an electrophoresis sample but containing a standard substance to be compared with the electrophoresis sample.
  • a medium-equipped holding body has a medium holding surface having a belt shape, and holds the first-dimensional electrophoresis medium along the medium holding surface. It is characterized by having.
  • a two-dimensional electrophoresis apparatus includes a pair of electrodes for first-dimensional electrophoresis, a cathode and an anode for second-dimensional electrophoresis, A two-dimensional electrophoresis apparatus that contains a molecular weight standard as a reference material for comparison of electrophoresis samples in one area and a medium containing the electrophoresis sample in the other area.
  • the pair of electrodes for the first-dimensional electrophoresis used as an electrophoresis medium is configured to apply an electric field to a region that does not contain the molecular weight standard in the first-dimensional electrophoresis medium.
  • the cathode and anode for electrophoresis are characterized in that an electric field is applied to the entire region of the first-dimensional electrophoresis medium.
  • a two-dimensional electrophoresis method includes a preparation step of introducing an electrophoresis sample into the first-dimensional electrophoresis medium, and a first-dimension following the preparation step.
  • a first-dimensional electrophoresis step in which an electric field is applied to the electrophoretic medium to perform the first-dimensional electrophoresis, and the first-dimensional electrophoretic medium after the first-dimensional electrophoretic step are attached to the end of the second-dimensional electrophoretic medium.
  • a second-dimensional electrophoresis step of performing a second-dimensional electrophoresis by applying an electric field to the second-dimensional electrophoresis medium in a contacted state is a preparation step of introducing an electrophoresis sample into the first-dimensional electrophoresis medium, and a first-dimension following the preparation step.
  • a jig includes: A jig for producing the first-dimensional electrophoretic medium, An introduction substance holding unit for holding an introduction substance containing a standard substance; A medium holding unit that holds the first-dimensional electrophoretic medium before the standard substance is introduced at a position adjacent to the introduction substance holding unit is provided.
  • another two-dimensional electrophoresis method introduces an electrophoresis sample into a partial region of the first-dimensional electrophoresis medium using the jig.
  • Example 1 Production of carrier with gel (introduction of molecular weight marker)] Supplied by the automatic two-dimensional electrophoresis apparatus Auto2D (Sharp Manufacturing System Co., Ltd.) in which a dry band-shaped IPG gel (52 mm length) is formed on the end surface of the chip as the holder 40 with the gel described in the first embodiment.
  • the IEF chip used was used.
  • the cathode and anode of the first-dimensional electrophoresis are separated by 48 mm, and when the IPG gel of the IEF chip is set, the distance between the electrodes is increased by 2 mm at both ends of the IPG gel. There will be an outside area. Therefore, 0.5 ⁇ l of molecular weight marker solution (BenchMark® Fluorescent® Protein® Standard, Life Technologies) was dropped onto the region using a pipettor. After dropping, the dropping area was dried by leaving it in a vacuum desiccator for about 1 hour. This dripping region is the marker introduction region 2.
  • molecular weight marker solution BenchMark® Fluorescent® Protein® Standard, Life Technologies
  • Example 2 Preparation of IPG gel containing molecular weight marker
  • a separation medium having a gradient in pH an IPG gel having a pH of 4-10 is prepared on a holding sheet (gel bond film: manufactured by Lonza), and is left to stand in a vacuum desiccator overnight to be dried to form a sheet-like IPG gel.
  • a holding sheet gel bond film: manufactured by Lonza
  • the marker liquid storage portion 102 of the marker introduction jig 100 shown in FIG. 4 is filled with a molecular weight marker (BenchMark FluorescentteProtein Standard: Invitrogen), and the sheet-like IPG gel is perpendicular to the pH gradient direction. It was immersed from the acid side gel end face and allowed to stand for about 1 hour.
  • a molecular weight marker BenchMark FluorescentteProtein Standard: Invitrogen
  • the sheet-like IPG gel was taken out from the marker introduction jig 100 and left in a vacuum desiccator for about 1 hour to dry the immersion area.
  • the sheet-like IPG gel was cut into strips in the pH gradient direction. Thereby, the IPG gel which introduce
  • Example 3 Two-dimensional electrophoresis using IPG gel containing molecular weight marker
  • the IPG gel prepared in Example 1 was evaluated based on the manufacturer's standard method using an automatic two-dimensional electrophoresis apparatus Auto2D (Sharp Manufacturing System Co., Ltd.).
  • Mouse liver samples pre-fluorescently labeled (IC5-OSu: Dojindo Chemical Co., Ltd.) gel swelling solution (urea, thiourea, 3- (3-cholamidepropyl) dimethylammonio-1-propanesulphonate (CHAPS), dithiothreitol (DTT), amphoteric
  • IC5-OSu Dojindo Chemical Co., Ltd.
  • gel swelling solution urea, thiourea, 3- (3-cholamidepropyl) dimethylammonio-1-propanesulphonate (CHAPS), dithiothreitol (DTT), amphoteric
  • CHAPS dimethylammonio-1-propanesulphonate
  • DTT dithiothreitol
  • amphoteric A sample solution was prepared by mixing with an electrolyte (including Amphoryte).
  • the sample solution was brought into contact with the isoelectric focusing region 3 (FIGS. 2 and 3) of the IPG gel prepared in Example 1 for 30 minutes to introduce the sample into the IPG gel. For 5 minutes to swell the gel. Then, it set to automatic two-dimensional electrophoresis apparatus Auto2D, and the isoelectric focusing was performed. Before starting electrophoresis, it was confirmed that the marker introduction region of the IPG gel was located outside the isoelectric point electrodes.
  • Second dimension electrophoresis was performed at a constant 20 mA for 35 minutes.
  • a band of a molecular weight marker could be confirmed in a portion surrounded by a broken line. Moreover, the spot of the mouse liver sample could be confirmed in the other part. In addition, in the mouse liver sample applied between the isoelectric electrodes, there are very few spots due to the mixing of molecular weight markers, and it is confirmed that the molecular weight markers applied between the electrodes are not mixed between the electrodes. I was able to.
  • Example 4 Two-dimensional electrophoresis using an IPG gel in which a molecular weight marker and a sample are simultaneously introduced
  • the solution chip supplied by the automatic two-dimensional electrophoresis apparatus Auto2D is used as the simultaneous introduction jig 500 in FIG. 12, and the sample introduction groove (in the sample solution recess 503 in FIG. 12) is used.
  • a chamber of 0.5 ⁇ l of solution (corresponding to the marker solution dent 502 in FIG. 12) was prepared at both ends outside the electrode.
  • the molecular weight marker of the molecular weight marker liquid in the chamber is adsorbed to the IPG gel at the same time when the sample liquid sample stored in the sample introduction groove (corresponding to the sample liquid depression 503 in FIG. 12) is introduced, Since the molecular weight marker is located between the electrodes, the molecular weight marker is held in the IPG gel without moving to the second-dimensional electrophoresis.
  • the sample was prepared by mixing a mouse liver sample previously fluorescently labeled (IC5-OSu: Dojin Chemical) with a gel swelling solution.
  • fluorescently labeled BenchMark® Fluorescent® Protein® Standard (Life Technologies) was used.
  • the sample and the molecular weight marker were introduced into the IPG gel for 30 minutes using the above jig, and the IPG gel was immersed in a gel swelling solution for 5 minutes to perform gel swelling, followed by isoelectric focusing.
  • Second dimension electrophoresis was performed at a constant 20 mA for 35 minutes.
  • a band of a molecular weight marker could be confirmed in a portion surrounded by a broken line. Moreover, the spot of the mouse liver sample could be confirmed in the other part.
  • Example 5 Production of carrier with gel (introduction of fluorescently labeled peptide)] Similar to Example 1 above, the isoelectric point of the dried belt-like IPG gel formed on the end face of the IEF chip supplied by the automatic two-dimensional electrophoresis apparatus Auto2D (Sharp Manufacturing System Co., Ltd.) To the electrophoresis region 3 (FIG. 1), 1-2 ⁇ l of the fluorescence-labeled peptide solution was dropped using a pipetter. This fluorescently labeled peptide functions as an isoelectric point marker.
  • the dropping region was dried to prepare a gel-attached holding body in which a fluorescently labeled peptide was previously introduced into a band-shaped IPG gel.
  • the isoelectric focusing can be performed by adsorbing the sample to the isoelectric focusing region 3 in the stage where the isoelectric focusing is performed on the holder with the gel.
  • the present invention can be suitably used for a two-dimensional electrophoresis apparatus.

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Abstract

　Afin de simplifier les actions de la personne réalisant une électrophorèse bidimensionnelle et de permettre la mise en œuvre de l'électrophorèse bidimensionnelle de haute reproductibilité, dans un mode de réalisation de la présente invention, une région (2), dans laquelle a été introduit un marqueur de poids moléculaire prédéterminé, est incluse sur une partie d'extrémité d'un gel de focalisation isoélectrique (1), et au cours de l'électrophorèse à focalisation isoélectrique, aucun champ électrique n'agit sur la région dans laquelle est introduit le marqueur (2), et au cours d'une seconde électrophorèse dimensionnelle, le marqueur de poids moléculaire, de la région (2) dans laquelle celui-ci est introduit, migre vers un gel (424) dans la technique SDS-PAGE, avec un échantillon qui a été soumis à l'électrophorèse à focalisation isoélectrique.
PCT/JP2014/079040 2013-11-01 2014-10-31 Milieu d'électrophorèse unidimensionnelle pour électrophorèse bidimensionnelle, support sur lequel est maintenu le milieu, dispositif d'électrophorèse bidimensionnelle utilisant le milieu, procédé d'électrophorèse bidimensionnelle utilisant le milieu et gabarit Ceased WO2015064736A1 (fr)

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JP2000249684A (ja) * 1999-03-02 2000-09-14 Sentan Kagaku Gijutsu Incubation Center:Kk 二次元分離方法
JP2007064848A (ja) * 2005-08-31 2007-03-15 Sharp Corp 自動化２次元電気泳動装置および装置構成器具
JP2008164319A (ja) * 2006-12-27 2008-07-17 National Institute Of Advanced Industrial & Technology 電気泳動用乾燥媒体への試料の導入方法及びそのための器具
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JP2000249684A (ja) * 1999-03-02 2000-09-14 Sentan Kagaku Gijutsu Incubation Center:Kk 二次元分離方法
JP2007064848A (ja) * 2005-08-31 2007-03-15 Sharp Corp 自動化２次元電気泳動装置および装置構成器具
JP2008164319A (ja) * 2006-12-27 2008-07-17 National Institute Of Advanced Industrial & Technology 電気泳動用乾燥媒体への試料の導入方法及びそのための器具
JP2013040792A (ja) * 2011-08-11 2013-02-28 Sharp Corp 電気泳動ゲルチップならびにその製造方法および製造キット

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