WO2016190321A1 - Electrophoresis gel, electrophoresis kit, electrophoresis device and electrophoresis method - Google Patents

Abstract

An electrophoresis gel which is composed of a polyacrylamide gel having a pH of more than 7.0 but 8.0 or less. An electrophoresis method which comprises a separation step wherein a sample is separated by means of electrophoresis that uses a polyacrylamide gel having a pH of more than 7.0 but 8.0 or less.

Description

Electrophoresis gel, electrophoresis kit, electrophoresis apparatus, and electrophoresis method

The present invention relates to an electrophoresis gel, an electrophoresis kit, an electrophoresis apparatus, and an electrophoresis method.

To date, the relationship between various diseases and biopolymers is becoming clear after the human genome project. In particular, proteins, one of the biopolymers, are directly involved in the functions of living cells, organs, and organs, and are largely due to differences in amino acid sequence and three-dimensional structure, chemical modifications such as sugar chains and phosphorylation. It is beginning to become clear that it can cause various diseases.

In this situation, many proteome analyzes are being conducted. The proteome means a specific cell, organ, and the whole protein produced by translation in the organ. Examples of the analysis include protein profiling and functional analysis. In particular, it is known that proteins synthesized in vivo after protein translation are controlled by post-translational modifications such as phosphorylation, and information on chemical modification of proteins will be available in the future. It can be one of the important items in proteome analysis. Therefore, a method for separating and detecting a sample containing a plurality of proteins with high accuracy is regarded as important, and development of an apparatus for that purpose is being promoted.

Currently, useful protein separation methods include gel electrophoresis, capillary electrophoresis, and liquid chromatography, but gel electrophoresis is generally widely used because of its simplicity and high resolution. .

Patent Document 1 discloses a system for gel electrophoresis including an electrophoresis gel having two ends, wherein the gel is a polyacrylamide gel including a neutral gel buffer, and one end of the gel is an anode. The other end is in contact with the cathode buffer solution, the gel buffer solution contains bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), and the cathode buffer comprises 3 Describes a system comprising-(N-morpholino) propanesulfonic acid (MOP) or 2- (N-morpholino) ethanesulfonic acid (MES), and wherein the electrophoresis gel is precast and suitable for storage Has been.

Japanese Patent Publication “JP 2007-263976 A (published on Oct. 11, 2007)”

In gel electrophoresis, it is preferable to improve the moving speed of the sample in the electrophoresis gel because the time required for electrophoresis can be shortened. In particular, a technique for improving the moving speed of a polymer sample is very useful in an electrophoresis gel used for the discharge transfer type electrophoresis described later.

The present invention has been made in view of the above problems, and has as its main object to provide a novel technique for improving the moving speed of a sample in an electrophoresis gel.

The electrophoresis gel according to one embodiment of the present invention is composed of a polyacrylamide gel having a pH greater than 7.0 and less than or equal to 8.0.

The electrophoresis method according to one embodiment of the present invention includes a separation step of separating a sample by electrophoresis using a polyacrylamide gel having a pH greater than 7.0 and less than or equal to 8.0.

According to one embodiment of the present invention, the moving speed of the sample in the electrophoresis gel can be improved.

It is a side sectional view showing typically the schematic structure of the electrophoresis device concerning one embodiment of the present invention. It is a side sectional view showing typically the schematic structure of the electrophoresis device concerning one embodiment of the present invention. It is a figure which shows the photograph of the electrophoresis result in an Example. It is a figure which shows the photograph of the electrophoresis result in an Example. It is a figure which shows the photograph of the electrophoresis result in an Example. It is a figure which shows the graph of the electrophoresis result in an Example. It is a figure which shows the photograph of the electrophoresis result in an Example.

[Embodiment 1]
(Electrophoresis gel)
In one embodiment, the present invention provides an electrophoresis gel. In this specification, “electrophoresis gel” refers to a gel used in gel electrophoresis.

In one embodiment, the electrophoresis gel according to the present invention comprises a polyacrylamide gel, and the pH of the polyacrylamide gel may be, for example, 6.0 or more and 8.8 or less, but is greater than 7.0 and 8. It is more preferably 0 or less, more preferably greater than 7.0 and 7.7 or less, and particularly preferably greater than 7.0 and 7.5 or less. Alternatively, it may be greater than 7.2 and not greater than 8.0, greater than 7.2 and not greater than 7.7, and greater than 7.2 and not greater than 7.5. In one aspect, the pH of the electrophoresis gel or polyacrylamide gel can be the pH of the gel buffer contained in the electrophoresis gel. When the pH of the polyacrylamide gel satisfies the above conditions, the moving speed of the sample in the electrophoresis gel can be improved.

In addition, the% T of the polyacrylamide gel can be, for example, 5.0% T or more and 20.0% T or less, and more preferably 6.5% T or more and 12.5% T or less. In the present specification, “% T” indicates the content (g / mL) of acrylamide contained in the polyacrylamide gel. The electrophoresis gel may have a structure comprising a separation gel for separating a sample and a concentration gel for concentrating the sample. In this case,% T of the concentration gel is% of the separation gel. It is preferable to make it smaller than T.

The% C of the polyacrylamide gel can be, for example, 2.6% C or less, but is preferably less than 2.4% C, less than 2.2% C, and less than 2.0% C. 1.5% C or less, 1.0% C or less, or 0.5% C or less is particularly preferable. Moreover, the lower limit of% C of the polyacrylamide gel is not particularly limited, but may be, for example, 0.1% C or 0.2% C. When% C of the polyacrylamide gel satisfies the above conditions, the moving speed of the sample in the electrophoresis gel can be further improved. As will be described later, this becomes remarkable when the cathode buffer contains a leading ion selected from the group consisting of tris and bicine as the leading ion.

In this specification, “% C” indicates the content (g / g) of the acrylamide crosslinking agent in the acrylamide contained in the polyacrylamide gel. That is, it can be said that a polyacrylamide gel of less than 2.4% C contains an acrylamide crosslinking agent of less than 2.4% by weight of the acrylamide contained in the polyacrylamide gel.

In one embodiment, such an electrophoresis gel is prepared by adding a acrylamide monomer and an acrylamide cross-linking agent to a pH adjusted gel buffer to prepare a gel solution, and injecting the gel solution into a gel preparation container. Can be prepared by polymerizing an acrylamide monomer and an acrylamide cross-linking agent. For the polymerization of the acrylamide monomer and the acrylamide crosslinking agent, a polymerization accelerator may be appropriately added to the gel solution.

The gel buffer is not particularly limited as long as it is an electrolytic solution containing a buffering agent. For example, Bis-Tris (bis (2-hydroxyethyl) -amino-tris (hydroxymethyl) methane), MOPS (3 (N-morpholino) It preferably contains a known buffer used for electrophoresis gels such as propanesulfonic acid) and HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), and contains Bis-Tris. It is particularly preferable. When the gel buffer contains Bis-Tris, the buffer region can suitably have a buffer region in the range of 7.0 to 8.0. The pH of the gel buffer can be adjusted to a desired range using an appropriate acid such as hydrochloric acid, sulfuric acid, acetic acid, boric acid, phosphoric acid, and glycolic acid.

An acrylamide cross-linking agent is a molecule that reacts with an acrylamide monomer to form a cross-link in the process of polymerization of the acrylamide monomer. The acrylamide cross-linking agent may be any known acrylamide cross-linking agent, such as N, N′-methylene-bis-acrylamide (bisacrylamide), ethylene diacrylate (ED), diallyl-tartaric acid diamide (DATD), and Examples thereof include dihydroxyethylene-bisacrylamide (DHEBA), and bisacrylamide is more preferable. In addition, what is necessary is just to adjust the quantity of the acrylamide monomer and acrylamide crosslinking agent which are added to a gel buffer so that it may match% T and% C mentioned above.

Examples of the polymerization accelerator include, but are not limited to, ammonium persulfate (APS), N, N′-tetramethylenediamine (TEMED), riboflavin, and β-dimethylaminopropionitrile. A combination of APS and TEMED is preferably used. For example, the polymerization accelerator is preferably added to the gel solution immediately before use of the electrophoresis gel.

Further, additional reagents or additives may be added to the gel solution. Although it does not specifically limit as a container for gel preparation, For example, the insulating plates 11 * 12 overlapped through the spacer as shown in FIG. 1 can be used. A comb for forming a well for loading a sample may also be used. As the size of the container, one having a size corresponding to the type, amount, etc. of the sample to be separated can be appropriately used.

In one embodiment, in an electrophoresis method using an electrophoresis gel, a sample is loaded at a specific position (for example, one end) of the electrophoresis gel, and a voltage is applied across the electrophoresis gel. By moving (electrophoresis) the biomolecule (for example, protein) contained in the sample in the electrophoresis gel with a different migration rate depending on each molecular weight, charge or both (separation step), Biomolecules can be separated into bands at different positions on the movement path in the electrophoresis gel.

The sample to be separated is not particularly limited. For example, a preparation from a biological material (for example, an individual organism, a body fluid, a cell line, a tissue culture, or a tissue fragment), or a commercially available reagent containing a biomolecule can be used. In particular, it may contain proteins or nucleic acids.

The method for applying a voltage to the electrophoresis gel is not particularly limited. For example, using an electrophoresis buffer (a cathode buffer and an anode buffer), one end of the electrophoresis gel is brought into contact with the cathode buffer, and the other of the electrophoresis gel is applied. The voltage can be applied to the electrophoresis gel by bringing the end into contact with the anode buffer and applying a voltage between the cathode disposed in the cathode buffer and the anode disposed in the anode buffer.

The electrophoresis buffer may be an electrolyte solution containing a buffer, and may have the same or different composition between the cathode buffer and the anode buffer. Examples of buffers that can be included in the electrophoresis buffer include Bis-Tris, MOPS, MES (2 (N-morpholino) ethanesulfonic acid), HEPES, Tris / glycine, acetic acid, sodium carbonate, CAPS, Tris / boric acid / Known buffering agents used for electrophoresis gels such as EDTA, Tris / acetic acid / EDTA, MOPS, phosphoric acid, Tris / Tricine, and the like are mentioned, and Bis-Tris is particularly preferable. Moreover, the electrophoresis buffer may contain a surfactant such as SDS.

Also, the electrophoresis buffer can further contain leading ions and trailing ions. Leading ions are substances that move the fastest in an electrophoresis gel during electrophoresis. When this moving speed is slow, it is difficult to separate low molecules. Trailing ions are substances that flow behind the sample during electrophoresis. The leading ions and trailing ions function by being electrophoresed together with the sample during electrophoresis, and need only be contained in the cathode buffer, and may not be contained in the anode buffer.

As the leading ion, for example, a known leading ion such as HCl may be used, but preferably the pH of the polyacrylamide gel is 7.0 by using a leading ion selected from the group consisting of tris and bicine. When it is larger and is 8.0 or less, a low molecular weight sample can be suitably separated.

As the trailing ions, for example, known trailing ions such as MOPS, MES, and glycine can be used, but it is more preferable to use trailing ions selected from the group consisting of MOPS and MES.

(Electrophoresis kit)
Thus, in one embodiment, the present invention provides an electrophoresis kit for electrophoresis. The electrophoresis kit can include an electrophoresis gel, a cathode buffer, and an anode buffer. The electrophoresis kit may further include other instruments and reagents used for electrophoresis, a document such as instructions, or a recording medium containing the document. Moreover, in one Embodiment, an electrophoresis kit can be integrated in the electrophoresis apparatus demonstrated below.

(Electrophoresis device)
In one embodiment, the electrophoresis gel and electrophoresis kit described above may be incorporated into an electrophoresis apparatus. In one embodiment, an electrophoresis apparatus according to the present invention includes the above-described electrophoresis kit, a cathode buffer tank in which a cathode buffer is stored and a cathode is disposed, an anode buffer tank in which an anode buffer is stored and an anode is disposed. The electrophoretic device may be provided with a gel holding unit that holds the electrophoresis gel so that one end thereof is in contact with the cathode buffer and the other end is in contact with the anode buffer.

FIG. 1 is a side sectional view showing an example of a schematic configuration of an electrophoresis apparatus according to an embodiment of the present invention. As shown in FIG. 1, the electrophoresis apparatus 10 includes an electrophoresis gel 5, insulating plates (gel holding units) 11 and 12, a cathode buffer tank 13 and an anode buffer tank 14, an anode 15 and a cathode 16.

The cathode buffer tank 13 stores a cathode buffer, and a cathode 16 is disposed therein. The anode buffer tank 14 stores an anode buffer, and an anode 15 is disposed therein. The insulating plates 11 and 12 have an upper end opened in the cathode buffer tank 13 and a lower end opened in the anode buffer tank 14 so that the electrophoresis gel 5 is in contact with the cathode buffer at one end, One end is held in contact with the anode buffer.

The anode 15 and the cathode 16 are made of a conductive material such as metal. As a material for forming the anode 15 and the cathode 16, for example, platinum is preferable from the viewpoint of suppressing ionization of the electrode.

Then, the sample can be separated by electrophoresis by loading (introducing) the sample into the electrophoresis gel 5 from the upper end of the electrophoresis gel 5 and passing a current between the anode 15 and the cathode 16. In addition to the biomolecule to be analyzed, it is preferable to add a visible molecular weight marker for confirming the progress of electrophoresis to the sample. In addition, the value of the current flowing between the anode 15 and the cathode 16 is not particularly limited, but is preferably 50 mA or less, and more preferably 20 mA or more and 30 mA or less. The current value may be controlled to be constant, the voltage may be controlled to be constant, or the current / voltage may be controlled in other manners.

After electrophoresis, the electrophoresis gel 5 from which the sample has been separated is removed from the electrophoresis apparatus 10 and subjected to subsequent processing for analyzing biomolecules (for example, transfer to a transfer membrane and staining or immune reaction, or gel It can be used for excision and recovery of the target protein.

[Embodiment 2]
Another embodiment (Embodiment 2) of the present invention will be described below with reference to the drawings.

The present invention is not limited to the configuration in which electrophoresis is performed in the manner described in the first embodiment, and can be applied to various configurations as long as gel electrophoresis is performed. For example, in one embodiment, the present invention separates a sample by electrophoresis, discharges the separated sample from the discharge part in the anode buffer tank, and moves the transfer film in contact with the discharge part to move the sample. The present invention can be applied to electrophoresis in which a sample is transferred to the transfer film (hereinafter referred to as “discharge transfer type electrophoresis”).

In one embodiment, the discharge transfer type electrophoresis is separated in the electrophoresis gel by moving the transfer film while facing the end in the anode buffer tank of the electrophoresis gel. It can be carried out by using an electrophoresis apparatus provided with a transfer part for transferring the sample discharged from the end part in the tank to the transfer film.

Such a transfer part is not limited to this, but, for example, a transfer film holding part that holds the transfer film facing the end in the anode buffer tank of the electrophoresis gel, and the transfer film holding This can be realized by a drive mechanism that moves the part. Alternatively, the transfer film may be realized by a roller-type paper feed mechanism that moves while facing the end of the electrophoresis gel in the anode buffer tank.

Here, in the discharge transfer type electrophoresis, it is necessary to further improve the electrophoresis speed of the polymer as compared with general electrophoresis. This is because in general electrophoresis, the biomolecules in each sample do not need to migrate to the tip of the electrophoresis gel, and in particular, the polymer remains on the back side of the electrophoresis gel even after electrophoresis. There are many cases. However, in electrophoretic electrophoresis, it is necessary to discharge all biomolecules in the sample containing the polymer from the tip of the electrophoresis gel. For this reason, there is a possibility that the time required for electrophoresis may be lengthened. Therefore, it is very important to improve the electrophoresis speed of the polymer in the discharge transfer type electrophoresis. According to the electrophoresis gel of the present invention, since the electrophoresis speed of the polymer can be improved, it can be suitably applied to the discharge transfer type electrophoresis.

FIG. 2 is a side sectional view showing another example of the schematic configuration of the electrophoresis apparatus according to the embodiment of the present invention. As shown in FIG. 2, the electrophoresis apparatus 100 separates the sample by electrophoresis, discharges the separated sample from the discharge portion, and moves the transfer film in contact with the discharge portion to move the sample. An electrophoretic apparatus for performing transfer-type electrophoresis that transfers a sample to the transfer film, including a clamp (transfer unit, transfer film holding unit) 20, a clamp (transfer unit, transfer film holding unit) 21, and a clamp frame (transfer) Part, transfer film holding part) 22, carrier (transfer part, driving mechanism) 23, anode buffer tank 30, table 31, cathode buffer tank 40, gel holding part 50, motor (transfer part, driving mechanism) 62, ball screw (transfer) Part, drive mechanism) 63, guide shaft (transfer part, drive mechanism) 64, shaft holder (transfer part, drive mechanism) 65, guide pole (transfer part, drive mechanism) 66, Control portion (transfer portion, the drive mechanism) and a 68. Further, although not shown for the sake of explanation, for the sake of safety, a lid that covers the whole during operation is further provided.

Here, the gel holding unit 50 houses the electrophoresis gel 52 and has a first opening (discharge unit) 50 a that opens into the anode buffer tank 30 and a second opening 50 b that opens into the cathode buffer tank 40. is doing. Further, the transfer film 1 is disposed in the anode buffer tank 30 so as to face the first opening 50a. An anode 32 is disposed in the anode buffer tank 30, and a cathode 41 is disposed in the cathode buffer tank 40.

For this reason, in the electrophoresis apparatus 100, the cathode buffer tank 40 is filled with the cathode buffer, and the anode buffer tank 30 is filled with the anode buffer, whereby the cathode 41 in the cathode buffer tank 40 and the anode 32 in the anode buffer tank 30 are formed. The two tanks are electrically connected via the electrophoresis buffer, the electrophoresis gel 52, and the transfer film 1. That is, the electrophoresis apparatus 100 separates the sample introduced from the second opening 50b by the electrophoresis gel 52 by applying a voltage between the cathode 41 and the anode 32, and separates the separated components into the first. This is an apparatus for discharging from the opening 50a and adsorbing it to the transfer film 1.

(Anode and cathode)
The anode 32 is disposed in the anode buffer tank 30, and the cathode 41 is disposed in the cathode buffer tank 40. Regarding the electrode arrangement, the anode 32 is arranged in the anode buffer tank 30 and the cathode 41 is not particularly limited as long as it is arranged in the cathode buffer tank 40. For example, the cathode 41, the first opening 50a and the anode 32 may be arranged on a substantially straight line. If the transfer film 1 is arranged in such an arrangement as shown in FIG. 2, the lines of electric force passing through the first opening 50a become substantially perpendicular to the transfer film 1, thereby improving the accuracy of sample adsorption. obtain.

Further, the anode 32 is preferably arranged away from the transfer film 1. Thereby, it is possible to suppress the bubbles generated from the anode 32 from adversely affecting the adsorption of the separation component to the transfer film 1.

The anode 32 and the cathode 41 may be used by being connected to the control unit 68 or may be used by being connected to an external power supply (DC power supply device), for example. When used by connecting to an external power supply, after setting the time, current and voltage to the power supply, the electrophoretic device 100 can be started by operating the control unit 68 simultaneously with the operation of the power supply. That's fine.

(Anode buffer tank and cathode buffer tank)
The anode buffer tank 30 and the cathode buffer tank 40 are insulating containers that retain the electrophoresis buffer. The cathode buffer tank 40 is provided above the anode buffer tank 30. In this embodiment, the anode buffer tank 30 is fixed on the table 31, and the cathode buffer tank 40 is fixed to the anode buffer tank 30, but the present invention is not limited to this configuration.

As will be described in detail later, guides 33 and 34 for supporting the transfer film 1 from the back surface of the transfer film 1 are provided in the movement path of the transfer film 1 at the bottom of the anode buffer tank 30.

(Gel holding part)
The gel holding unit 50 houses an electrophoresis gel 52 therein. In the present embodiment, the gel holding unit 50 stands up in a substantially vertical direction, the lower part thereof is arranged in the anode buffer tank 30, and the upper part thereof is arranged so that one side thereof is in contact with the cathode buffer tank 40. Thereby, the electrophoresis gel 52 is water-cooled by at least one of the anode buffer in the anode buffer tank 30 and the cathode buffer in the cathode buffer tank 40, and can be sufficiently cooled.

The gel holding unit 50 has a first opening 50 a that opens into the anode buffer tank 30 and a second opening 50 b that opens into the cathode buffer tank 40. Thus, the electrophoresis gel 52 faces the anode buffer tank 30 through the first opening 50a and faces the cathode buffer tank 40 through the second opening 50b. In this embodiment, the gel holding unit 50 is fixed to the cathode buffer tank 40 by a lock 42 provided in the cathode buffer tank 40, but the present invention is not limited to this configuration.

The gel holding part 50 may be composed of two insulating plates 51 and 53 formed of an insulator such as glass or acrylic. In one embodiment, the gel holding part 50 exposes the electrophoresis gel 52 by lacking a part of the insulating plate 53 in the second opening 50 b, thereby making it easy to sample the electrophoresis gel 52. Can be introduced.

Further, the first opening 50a of the gel holding unit 50 is formed of a conductive porous material (for example, a hydrophilic PVDF (Polyvinylidene difluoride) film, a hydrophilic PTFE (Polytetrafluoroethylene) film, etc.) including the periphery thereof. It may be covered with a covering portion. Accordingly, when the transfer film 1 is in contact with or pressed against the first opening 50a (when no distance is provided between the first opening 50a and the transfer film 1), the transfer film 1 is transferred when the transfer film 1 is conveyed. The frictional resistance and damage received from the gel holder 50 and the electrophoresis gel 52 can be reduced.

In addition, since the gel holding | maintenance part 50 stands up in the substantially vertical direction, the sample introduction amount can be increased compared with the structure in which the gel holding | maintenance part 50 is installed in the substantially horizontal direction. This is because it is difficult to change the depth of the well provided in the separation gel in the horizontal electrophoresis apparatus, but the depth of the well can be easily changed in the vertical electrophoresis apparatus. This is because the amount can be easily increased.

(Transfer film 1)
The transfer film 1 is preferably a sample adsorbing / holding body that allows the sample separated by the electrophoresis gel 52 to be stably stored for a long period of time and further facilitates subsequent analysis. The material of the transfer film 1 is preferably a material having high strength and high sample binding ability (weight that can be adsorbed per unit area). As the transfer film 1, a PVDF film or a nitrocellulose film is suitable when the sample is a protein. Note that the PVDF membrane is preferably hydrophilized in advance using methanol or the like. In addition to this, membranes conventionally used for protein, DNA and nucleic acid adsorption such as nitrocellulose membrane or nylon membrane can also be used.

The sample that can be separated and adsorbed in the electrophoresis apparatus 100 is not limited to these, but a preparation from a biological material (for example, a biological individual, a body fluid, a cell line, a tissue culture, or a tissue fragment), or A commercially available reagent etc. are mentioned. For example, a polypeptide or polynucleotide is mentioned.

The transfer film 1 is used in a state immersed in the anode buffer in the anode buffer tank 30.

In this embodiment, the transfer film 1 has a length that is used for one electrophoresis / transfer, in other words, a distance that moves in the anode buffer tank 30 in one electrophoresis / transfer. If you do. By configuring the transfer film 1 in this way, the operation of cutting the transfer film 1 is not required for each electrophoresis / transfer, and the usability of the electrophoresis apparatus 100 can be improved. Further, the lateral width of the transfer film 1 may be a length corresponding to the lateral width of the electrophoresis gel 52.

(Adjuster)
In the present embodiment, the transfer film 1 is used in a state of being held by an adjuster (transfer film holding unit) 2 so as to face the end of the electrophoresis gel 52 in the anode buffer tank 30. The adjuster 2 includes clamps 20 and 21 and a clamp frame 22, and is disposed inside the side wall of the anode buffer tank 30.

The clamp 20 includes an elastic body 20a and a pressing member 20b. The elastic body 20a is brought into contact with the back surface of the transfer film 1 (the surface opposite to the surface that is in contact with the first opening 50a), and is pressed by the pressing member 20b. The transfer film 1 is fixed by pressing the transfer film 1 against the elastic body 20 a from the surface side of the transfer film 1. In particular, the pressing member 20b is preferably configured to push the transfer film 1 into the elastic body 20a.

Similarly, the clamp 21 includes an elastic body 21a and a pressing member 21b. The elastic body 21a is in contact with the back surface of the transfer film 1 (the surface opposite to the surface in contact with the first opening 50a) and pressed. The transfer film 1 is fixed by pressing the transfer film 1 against the elastic body 20a from the surface side of the transfer film 1 by the member 21b. In particular, the pressing member 21b is preferably configured to press the transfer film 1 into the elastic body 21a.

Here, the function of the adjuster 2 will be described. The adjuster 2 is a structure that holds the transfer film 1, and is preferably configured to maintain a state where the tension of the transfer film 1 is as high as possible. This is because if the tension of the transfer film 1 is low (the transfer film is loosened), the transfer film 1 can be brought into close contact with the first opening 50a even if the transfer film 1 is brought into contact with the first opening 50a of the gel holding unit 50. Because it is difficult. As will be described below, the adjuster 2 according to the present embodiment suitably maintains the tension of the transfer film 1, and can easily bring the transfer film 1 into close contact with the first opening 50a.

When the transfer film 1 is held by the adjuster 2, that is, when the transfer film 1 is fixed by the clamps 20 and 21, when the transfer film 1 contacts the first opening 50a of the gel holding unit 50, the transfer is performed. The film 1 is pushed down by the first opening 50a, and the elastic bodies 20a and 21a are pushed down by the pushed-down transfer film 1 inside the fixed positions of the clamps 20 and 21. At this time, since the repulsive force from the elastic bodies 20a and 21a is applied to the transfer film 1, the tension of the transfer film 1 can be maintained. Thereby, the transfer film 1 can be suitably adhered to the first opening 50a.

The material of the elastic bodies 20a and 21a has a softness that allows the pressing members 20b and 21b and the transfer film 1 to be fitted therein, and the transfer film 1 can be fixed in pairs with the pressing members 20b and 21b. Although it will not specifically limit if it is an elastic body which can be used, For example, elastomers, such as silicon sponge, urethane rubber, chloroprene rubber, and fluororubber, can be used conveniently.

The clamp frame 22 is a shaft member that connects the clamps 20 and 21, and connects the clamps 20 and 21 with a predetermined distance therebetween. Thereby, when both ends of the transfer film 1 are fixed by the clamps 20 and 21, the transfer film 1 can be stretched without loosening along the moving direction. Thereby, it can suppress that a transcription | transfer result shakes by the looseness of the transfer film 1, and can improve a measurement sensitivity. Further, the tension applied to the transfer film 1 conveyed along with the movement of the clamp 20 can be made constant. Therefore, the sample can be more suitably transferred to the transfer film 1 without unevenness.

The clamp frame 22 is disposed at a position sandwiching the transfer film 1 from the side in the movement direction, and thereby, the front surface (surface facing the first opening 50a) and the back surface (opposite to the first opening 50a) of the transfer film 1. It can be avoided that the clamp frame 22 overlaps the side surface. Accordingly, it is possible to prevent the clamp frame 22 from inhibiting the transfer from the electrophoresis gel 52 to the transfer film 1 and the contact of other members to the back surface of the transfer film 1 (details will be described later). Further, fixing of the transfer film 1 by the clamps 20 and 21 is not inhibited.

The clamp frame 22 and the clamps 20 and 21 (excluding the elastic bodies 20a and 21a) are not limited to this, but may be made of a synthetic resin such as Teflon (registered trademark), acrylic resin, or PEEK resin.

(Arm part)
In the present embodiment, the adjuster 2 is incorporated in the arm portion. The arm portion moves the transfer film 1 and brings it into contact with the first opening 50a. In the present embodiment, the arm portion includes the adjuster 2, the carrier 23, and the guide pole 66 that are a series of connected members.

The guide pole 66 is a shaft member that is connected to a drive unit (shaft holder 65), which will be described later, and is disposed so as to pass outside the side wall of the anode buffer tank 30. The carrier 23 is a member that is connected to the guide pole 66, goes around the upper end of the side wall of the anode buffer tank 30, and is connected to the clamp 20.

As described above, the arm portion is connected to the inside of the side wall through the outside of the side wall of the anode buffer tank 30 from the position where it is connected to the driving unit, and around the upper end of the side wall.

Although the present invention is not limited, in this embodiment, the guide pole 66 extends outside the side wall of the anode buffer tank 30 to a position aligned with the upper end of the side wall. The carrier 23 is fitted to the guide pole 66 and extends inside the side wall across the upper end of the side wall of the anode buffer tank 30.

With this configuration, the carrier 23 can be easily attached to and detached from the drive unit. The guide pole 66 is disposed outside the side wall of the anode buffer tank 30 and does not interfere with various operations such as removal of the anode buffer tank 30 and electrode setting, which are performed as necessary. Therefore, various operations can be successfully performed by appropriately removing the carrier 23.

(Drive part)
The drive unit drives the arm unit in a substantially horizontal direction, and in the present embodiment, is constituted by a motor 62, a ball screw 63, a guide shaft 64, and a shaft holder 65.

The motor 62 rotates the ball screw 63. As the motor 62, a motor whose speed can be changed may be used, or a motor having a fixed speed may be used in combination with a gear. The ball screw 63 penetrates the shaft holder 65 and is screwed into the shaft holder 65. The guide shaft 64 passes through the shaft holder 65, and the shaft holder 65 is configured to be movable along the guide shaft 64. Then, when the motor 62 rotates the ball screw 63, the shaft holder 65 is driven in the X direction (substantially horizontal direction) in the figure. The shaft holder 65 is connected to the arm portion (guide pole 66), and thus, the drive portion can drive the arm portion in the X direction (substantially horizontal direction) in the drawing. Since the arm portion holds the transfer film 1, the transfer film 1 moves in the X direction (substantially horizontal direction) in the figure.

However, the present invention is not limited to this, and the driving unit may be configured by another driving mechanism (for example, a belt, a gear, or the like) as long as the arm unit can be driven in a substantially horizontal direction. Good.

The drive unit is provided under the anode buffer tank 30. Accordingly, it is possible to prevent the buffer solution scattered from the anode buffer tank 30 from deteriorating the durability of the driving unit and the driving unit from interfering with various operations on the electrophoretic device 100.

(Control part)
The control unit 68 is a control panel that performs various controls of the electrophoresis apparatus 100 (control of the position of the arm unit, control of current and voltage applied to the anode 32 and the cathode 41, etc.). The control unit 68 may include a button and a switch for receiving an input from the user, a lamp for notifying the user of an operation state, a display unit, and the like.

(Sample electrophoresis and transcription)
Next, the flow of sample electrophoresis and transfer in the electrophoresis apparatus 100 will be described with reference to FIG. As shown in FIG. 2, at the time of electrophoresis and transfer of the sample, the transfer film 1 is held by the adjuster 2 in a state of being disposed at a position in contact with the first opening 50a. At this time, the transfer film 1 is supported from the back surface of the transfer film 1 (the side opposite to the gel holding unit 50) by guides 33 and 34 provided at the bottom of the anode buffer tank 30.

The guides 33 and 34 are provided at the bottom of the anode buffer tank 30 so as to support the transfer film in the moving path along which the transfer film 1 moves. The longitudinal directions of the guides 33 and 34 are orthogonal to the moving direction (X direction) of the transfer film 1 and are parallel to the longitudinal direction of the first opening 50a.

The gel holding unit 50 (on the first opening 50a side) abuts on the surface of the transfer film 1 (the gel holding unit 50 side), whereby the transfer film 1 is convex on the side opposite to the gel holding unit 50. It is bent like this. In this way, the transfer film 1 is supported by the guides 33 and 34, and the gel holding part 50 is pressed down and bent so as to protrude downward (opposite to the gel holding part 50). As a result, tension is applied to the transfer film 1, and the transfer film 1 can be brought into close contact with the first opening 50a. Thereby, transfer from the electrophoresis gel 52 to the transfer film 1 can be performed more suitably.

In particular, the guides 33 and 34 are formed on the bottom of the anode buffer tank 30 at positions facing the first opening 50a in pairs, so that the guides disposed on both sides of the gel holding unit 50 are formed. The transfer film 1 is supported by 33 and 34, and the gel holding part 50 is pressed down and bent so as to protrude downward (to the side opposite to the separation part). As a result, a uniform tension is applied to the transfer film 1, and the transfer film 1 can be evenly adhered to the first opening 50a. Thereby, transfer from the electrophoresis gel 52 to the transfer film 1 can be performed more suitably.

Note that, as described above, the clamp frame 22 is disposed at a position sandwiching the transfer film 1 from the side in the movement direction, so that the guides 33 and 34 do not prevent the transfer film 1 from being supported from the back surface.

Then, the sample is introduced into the electrophoresis gel 52 from the second opening 50 b of the gel holding unit 50. In the above state, the sample is separated by electrophoresis. The control unit 68 controls the motor 62 to set the position of the transfer film 1 as a start position, and allows current to flow between the anode 32 and the cathode 41 to start electrophoresis. The value of the current that flows between the anode 32 and the cathode 41 is not particularly limited, but is preferably 50 mA or less, and more preferably 20 mA or more and 30 mA or less. The current value may be controlled to be constant, the voltage may be controlled to be constant, or the current / voltage may be controlled in other manners.

The transfer film 1 is gradually moved in the X direction (substantially horizontal direction) by driving the arm unit (adjuster) by the driving unit in accordance with the progress of electrophoresis in the gel holding unit 50. The X direction is a direction orthogonal to the longitudinal direction of the first opening 50a. The moving speed of the transfer film 1 is not particularly limited, but can be a pace that moves 5 to 10 cm in 60 to 120 minutes, for example.

Then, the position of the sample discharged by electrophoresis (separated in the electrophoresis gel 52) from the first opening 50a according to the discharge timing in the transfer film 1 (the first opening at the discharge timing). Adsorbed at the position facing 50a). As a result, the separated sample is transferred to the transfer film 1.

After the transfer, the transfer membrane 1 can be collected and used for staining or immune reaction (blocking and antigen-antibody reaction in Western blotting). Thereafter, a separation pattern of components transferred to the transfer film 1 is detected by a fluorescence detector or the like. Such a fluorescence detector may be incorporated in the electrophoretic device 100, which can automate all the steps of electrophoresis, transfer, and detection.

[Summary]
The electrophoresis gel (5, 52) according to Embodiment 1 of the present invention is a polyacrylamide gel having a pH greater than 7.0 and not greater than 8.0.

According to the above configuration, the moving speed of the sample in the electrophoresis gel can be improved.

The electrophoresis gel according to aspect 2 of the present invention is the above-described aspect 1, wherein the polyacrylamide gel may contain an acrylamide cross-linking agent that is less than 2.4% by weight of acrylamide contained in the polyacrylamide gel. .

The electrophoresis gel which concerns on aspect 3 of this invention consists of polyacrylamide gel, Even if the said polyacrylamide gel contains the acrylamide crosslinking agent of less than 2.4 weight% of the acrylamide contained in the said polyacrylamide gel. Good.

According to the above configuration, the moving speed of the sample in the electrophoresis gel can be further improved. This effect is particularly remarkable when the cathode buffer used for electrophoresis contains a leading ion selected from the group consisting of tris and bicine.

In the electrophoresis gel according to aspect 4 of the present invention, in the above aspects 1 to 3, the polyacrylamide gel may contain bis (2-hydroxyethyl) -amino-tris (hydroxymethyl) methane.

According to the above configuration, electrophoresis can be suitably performed.

The electrophoresis kit according to aspect 5 of the present invention includes the electrophoresis gel according to aspects 1 to 4, a cathode buffer, and an anode buffer.

According to the above configuration, electrophoresis can be suitably performed.

In the electrophoresis kit according to aspect 6 of the present invention, in the above aspect 5, the cathode buffer may contain a leading ion selected from the group consisting of tris and bicine.

According to the above configuration, low molecules contained in the sample can also be suitably separated.

The electrophoresis kit according to aspect 7 of the present invention is the above-described aspect 5 or 6, wherein the cathode buffer includes bis (2-hydroxyethyl) -amino-tris (hydroxymethyl) methane and 3 (N-morpholino) propanesulfone. And a trailing ion selected from the group consisting of an acid and 2 (N-morpholino) ethanesulfonic acid.

According to the above configuration, electrophoresis can be suitably performed.

The electrophoresis apparatus (10, 100) according to Aspect 8 of the present invention includes the electrophoresis kit according to Aspects 5 to 7, and the cathode buffer tank (13, 41) in which the cathode buffer is stored and the cathode (16, 41) is disposed. 40), the anode buffer tank (14, 30) in which the anode buffer is stored and the anodes (15, 32) are arranged, and the electrophoresis gel, one end of which contacts the cathode buffer, And a gel holding part for holding the one end so as to be in contact with the anode buffer.

According to the above configuration, electrophoresis can be suitably performed.

The electrophoresis device according to Aspect 9 of the present invention is separated in the electrophoresis gel in Aspect 8 by moving the transfer film (1) while facing the other end of the electrophoresis gel. The transfer part (adjuster 2 (clamps 20 and 21 and clamp frame 22), the carrier 23, the motor 62, the ball screw 63, the guide shaft 64, and the shaft holder that transfers the sample discharged from the other end to the transfer film. 65, a guide pole 66, and a control unit 68).

According to the above configuration, it is possible to perform discharge transfer type electrophoresis by providing a transfer portion. Here, in the discharge transfer type electrophoresis, since it is necessary to perform electrophoresis to the tip of the electrophoresis gel, it is very important to improve the electrophoresis speed of the polymer. According to the present invention, since the electrophoresis speed of the polymer can be improved, the discharge transfer type electrophoresis can be suitably performed.

The electrophoresis method according to aspect 10 of the present invention includes a separation step of separating a sample by electrophoresis using a polyacrylamide gel having a pH greater than 7.0 and less than or equal to 8.0.

According to the above configuration, the moving speed of the sample in the electrophoresis gel can be improved.

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.

(Test 1)
A plurality of 1% C polyacrylamide gels having a pH changed in the range of 6.0 to 8.0 were prepared, and electrophoresis was performed under the same conditions. The effect of gel pH differences on electrophoresis was tested.

The polyacrylamide gel was prepared in a mini gel size of 70 mm in length and 1 mm in thickness. Bis-Tris buffer was used as the gel buffer. The final concentration of Bis-Tris was 375 mM. As the polymerization accelerator, ammonium persulfate (APS) and N, N′-tetramethylenediamine (TEMED) were used. Bisacrylamide was used as the acrylamide cross-linking agent. The separation gel had an acrylamide concentration of 10% T, and the concentrated gel had an acrylamide concentration of 4-5% T.

As an anode buffer and a cathode buffer, 50 mM Bis-Tris · HCl, 50 mM MOPS, 0.15% SDS aqueous solution was used. Electrophoresis was performed at a constant current of 40 mA for 30 minutes. As a sample, a commercially available colored molecular weight marker (Precision Protein 2 color standard, manufactured by Biorad Laboratories) was used.

3 (a) to 3 (j), the pH is 6.0, 6.4, 6.9, 7.0, 7.1, 7.2, 7.3, 7.5, 7. It is a photograph which shows the electrophoresis result when using a polyacrylamide gel of 7, 8.0. The sample was loaded on the upper end of the polyacrylamide gel and electrophoresed downward on the paper.

As shown in FIG. 3, when the pH of polyacrylamide is greater than 7.0 and less than or equal to 8.0 ((e) to (h) in FIG. 3), the pH of polyacrylamide is less than or equal to 7 (FIG. 3). (A) to (d)), the moving speed of the sample was remarkably improved.

When the pH is 8.0, the polyacrylamide gel becomes high resistance and generates heat, so the resolution of the band generated by loading the sample is deteriorated, but the moving speed of the sample is fast. It was confirmed.

(Test 2)
Subsequently, even when a 2.6% C polyacrylamide gel was used, it was tested whether or not the moving speed of the sample was significantly improved by increasing the pH of the polyacrylamide above 7.0.

As in Test 1, a plurality of types of polyacrylamide gels were prepared and subjected to electrophoresis under the same conditions, and the results were compared. (A) to (d) of FIG. 4 are respectively 1% C and pH 7.3, 1% C and pH 6.0, 2.6% C and pH 7.3, 2.6% C and pH 6.0. It is a photograph which shows the electrophoresis result when using a polyacrylamide gel. The conditions other than% C and pH of the polyacrylamide gel were the same as in Test 1.

As shown in FIG. 4, even when the% C of the polyacrylamide gel is other than 1% C, the pH of the polyacrylamide is higher than 7.0 and not higher than 8.0 (FIGS. 4A and 4C). )), The moving speed of the sample was remarkably improved as compared with the case where the pH of polyacrylamide was 7 or less (FIGS. 4B and 4D).

(Test 3)
Subsequently, it was examined how the difference in the composition of the electrophoresis buffer affects the electrophoresis when the pH of the polyacrylamide is higher than 7.0 and lower than 8.0.

As in Test 1, a plurality of types of polyacrylamide gels were prepared and subjected to electrophoresis under the same conditions except for the composition of the electrophoresis buffer, and the results were compared. The composition of the running buffer is 50 mM Bis-Tris · HCl, 50 mM MOPS, 50 mM Tris, 50 mM bicine, 0.15% SDS running buffer A, or 50 mM Bis-Tris · HCl, 50 mM MOPS, 0.15% SDS. Running buffer B was used.

FIGS. 5A to 5D show 2.6% when 1% C polyacrylamide gel and running buffer A are used, and 1% C polyacrylamide gel and running buffer B are used, respectively. It is a photograph which shows the electrophoresis result when 2.6% C polyacrylamide gel and electrophoresis buffer B are used when C polyacrylamide gel and electrophoresis buffer A are used. The pH of the polyacrylamide gel was 7.3 for all. Other conditions were the same as in Test 1.

As shown in FIG. 5, in any case where% C of the polyacrylamide gel is used, the electrophoresis buffer A is used ((a) and (c) of FIG. 5), and the electrophoresis buffer B is used (FIG. 5). (B) and (d)) were able to detect a low molecular weight protein marker (10 kDa) that could not be detected.

When electrophoresis buffer A is used, bicine and tris contained in electrophoresis buffer A function as (i) leading ions, (ii) pH of polyacrylamide is higher than 7.0, and 8.0. Unlike the case where only HCl ions act as leading ions by moving faster than HCl ions under the following conditions (when electrophoresis buffer B is used), the pH of polyacrylamide is greater than 7.0, Even in the case of 8.0 or less, it shows that a low molecular weight protein marker such as 10 kDa can be suitably separated.

(Test 4)
Subsequently, when the pH of polyacrylamide is higher than 7.0 and lower than 8.0, how the difference in% C of polyacrylamide gel and the difference in composition of electrophoresis buffer affect electrophoresis. Further details were tested.

Specifically, when the electrophoresis buffer A was used and when the electrophoresis buffer B was used, it was compared how the electrophoresis speed changes when using polyacrylamide gels having different% C. . Other conditions were the same as in Test 1.

FIG. 6 is a graph showing the movement distance in the polyacrylamide gel of protein markers of various molecular weights contained in the sample. 6A shows the case where the electrophoresis buffer A is used, and FIG. 6B shows the case where the electrophoresis buffer B is used. The vertical axis of each graph indicates the molecular weight of each protein marker, and the horizontal axis indicates the movement distance (relative degree) of each protein marker after electrophoresis. Each graph shows a case where 1% C polyacrylamide gel is used and a case where 2.6% C polyacrylamide gel is used.

As shown in FIG. 6, when running buffer A was used, a low molecular weight (10 kDa) sample could be separated. In addition, when the electrophoresis buffer A was used, the sample moving speed could be further improved by using a lower% C (1% C) polyacrylamide gel.

(Test 5)
Next, how the difference in% C of polyacrylamide gel affects electrophoresis when the pH of polyacrylamide is greater than 7.0 and less than 8.0 and electrophoresis buffer A is used. Further details were tested.

Specifically, in the case of using electrophoresis buffer A, it was compared how the electrophoresis speed changes when using polyacrylamide gels with different% C. The pH of the polyacrylamide gel was 7.3 for all. Other conditions were the same as in Test 1.

(A) to (d) in FIG. 7 are photographs showing the results of electrophoresis when using polyacrylamide gels with% C of 2.6, 1, 0.5, and 0.2, respectively.

As shown in FIG. 7, as the% C of the polyacrylamide gel decreases to 2.6% C, 1.0% C, 0.5% C, and 0.2% C, the moving speed of the sample is improved. It was. In particular, the migration rate of medium to high molecular weight protein markers of 25 kDa or more contained in the sample was improved. Almost no change was observed in the migration speed of the protein marker of less than 25 kDa. In the discharge transfer type electrophoresis, it was confirmed in this test that the middle molecular weight to high molecular weight protein marker was discharged quickly, and that the problem could be solved by lowering% C.

The present invention can be used for biomolecule analysis technology.

1 Transfer film 2 Adjuster (transfer section, transfer film holding section)
5 Electrophoresis gel 10 Electrophoresis device 20 Clamp (transfer part, transfer film holding part)
21 Clamp (transfer part, transfer film holding part)
20a / 21a elastic body 20b / 21b pressing member 22 clamp frame (transfer part, transfer film holding part)
23 Carrier (transfer section, drive mechanism)
30 Anode buffer tank 31 Table 32 Anode 33/34 Guide 40 Cathode buffer tank 41 Cathode 42 Lock 50 Gel holding part 50a First opening (discharge part)
50b Second opening 51/53 Insulating plate 52 Electrophoresis gel 62 Motor (transfer section, drive mechanism)
63 Ball screw (transfer section, drive mechanism)
64 Guide shaft (transfer section, drive mechanism)
65 Shaft holder (transfer section, drive mechanism)
66 Guide pole (transfer section, drive mechanism)
68 Control unit (transfer unit, drive mechanism)
100 Electrophoresis device

Claims (10)

1. Electrophoresis gel consisting of polyacrylamide gel with pH greater than 7.0 and less than or equal to 8.0.
2. The electrophoresis gel according to claim 1, wherein the polyacrylamide gel contains an acrylamide cross-linking agent of less than 2.4% by weight of acrylamide contained in the polyacrylamide gel.
3. Made of polyacrylamide gel,
   The polyacrylamide gel is an electrophoresis gel containing an acrylamide crosslinking agent of less than 2.4% by weight of acrylamide contained in the polyacrylamide gel.
4. The electrophoresis gel according to any one of claims 1 to 3, wherein the polyacrylamide gel contains bis (2-hydroxyethyl) -amino-tris (hydroxymethyl) methane.
5. An electrophoresis kit comprising the electrophoresis gel according to any one of claims 1 to 4, a cathode buffer, and an anode buffer.
6. The electrophoresis kit according to claim 5, wherein the cathode buffer contains a leading ion selected from the group consisting of Tris and bicine.
7. The cathode buffer is a tray selected from the group consisting of bis (2-hydroxyethyl) -amino-tris (hydroxymethyl) methane, 3 (N-morpholino) propanesulfonic acid and 2 (N-morpholino) ethanesulfonic acid. The electrophoresis kit according to claim 5 or 6, comprising ring ions.
8. An electrophoresis kit according to any one of claims 5 to 7,
   The cathode buffer is stored, a cathode buffer tank in which the cathode is disposed,
   The anode buffer is stored, and an anode buffer tank in which the anode is disposed,
   An electrophoresis apparatus, comprising: a gel holding unit that holds the electrophoresis gel such that one end thereof is in contact with the cathode buffer and the other end is in contact with the anode buffer.
9. Transfer that transfers the sample separated on the electrophoresis gel and discharged from the other end by moving the transfer film while facing the other end of the electrophoresis gel onto the transfer film The electrophoresis apparatus according to claim 8, further comprising a unit.
10. An electrophoresis method including a separation step of separating a sample by electrophoresis using a polyacrylamide gel having a pH greater than 7.0 and less than or equal to 8.0.

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