JP4590615B2 - Two-dimensional electrophoresis method - Google Patents

Description

  The present invention relates to a two-dimensional electrophoresis method and a kit for two-dimensional electrophoresis.

  Conventionally, in a two-dimensional electrophoresis method, a one-dimensional gel (meaning a gel used in the first-dimensional electrophoresis) and a two-dimensional gel (meaning a gel used in the second-dimensional electrophoresis) are separate apparatuses. Was being experimented with. For this reason, after completion of the first-dimensional electrophoresis, the gel used in the first-dimensional electrophoresis was taken out, and then the taken-out one-dimensional gel was joined to the two-dimensional gel. The removal and joining of the gel is done manually, so it is difficult to automate the device.In addition, a high degree of skill is required to properly join the two-dimensional gel, and the first-dimensional gel can be easily cut during operation. There was a problem of distraction and bending.

  Therefore, in recent years, in order to eliminate the adverse effects of taking out and joining the one-dimensional gel, the one-dimensional gel and the two-dimensional gel are supported on one support substrate with an interval (Patent Document 1). At the end of the electrophoresis, a method of injecting a third conductive gel during the interval to bring the one-dimensional gel and the two-dimensional gel into contact has been attempted. (Patent Documents 2 to 4)

  However, in the method of connecting the one-dimensional gel and the two-dimensional gel by injecting the conductive gel, it is necessary to inject and solidify the gelling solution for the connection when connecting the one-dimensional gel and the two-dimensional gel. In addition, there is a problem that complicated operations are required, such as temperature control for solidification and addition of reagents.

JP 58-193446 A Japanese Patent Laid-Open No. 61-288148 Japanese Patent Laid-Open No. 2-151758 JP-T-2004-510168

  The present invention is intended to perform the operations required for two-dimensional electrophoresis in a total and simple and quick manner. A single device can perform a two-dimensional electrophoretic connection operation of a one-dimensional gel and a two-dimensional gel quickly and easily. An object is to provide an electrophoresis method and a kit for two-dimensional electrophoresis.

The inventors of the present application have made extensive studies to solve the above-mentioned problems, and prepared the dried gel in the interval in advance, and poured the second swelling solution after the primary separation and before the secondary separation to swell the dried gel. Thus, it has been found that a one-dimensional gel and a two-dimensional gel can be interconnected quickly and easily.
That is, the present invention includes the following.

[1] Two-dimensional electrophoresis method including the following steps:
(1) A first electrophoretic separation medium in the form of a strip and a second electrophoretic separation medium are supported on a single support substrate at an interval from each other, and swell to form the first and second electrophoretic separation media. Providing a two-dimensional electrophoretic substrate, wherein a dry gel capable of connecting an electrophoretic separation medium is disposed at a position not in contact with the first electrophoretic separation medium during the interval;
(2) impregnating the first electrophoretic separation medium with a sample;
(3) applying an electric field to the first electrophoretic separation medium to primarily separate components in the sample;
(4) After the primary separation, a second swelling solution is added to the dried gel, and the first electrophoretic separation medium and the second electrophoretic separation medium are connected by the swollen gel. Swell the dried gel, and (5) both the first electrophoretic separation medium and the second electrophoretic separation medium are substantially in the longitudinal direction of the first electrophoretic separation medium. Applying an electric field in a vertical direction to the second electrophoretic separation medium to secondary-separate the primary separated components.
[2] The method according to [1], wherein the dry gel is disposed on the substrate at the interval.
[3] The dried gel is
In the step (1), the first electrophoretic separation medium and the second electrophoretic separation medium are disposed at positions that do not come into contact with each other,
The method according to [1] or [2], which is a gel that swells in the direction of the first electrophoretic separation medium and the direction of the second electrophoretic separation medium in the step (4).
[4] The dry gel is a gel contracted by drying a raw material gel, and the raw material gel has a size capable of connecting between the first electrophoretic separation medium and the second electrophoretic separation medium. The method according to any one of [1] to [3].
[5] The method according to [4], wherein the dry gel is obtained by drying the raw material gel while being compressed.
[6] The method according to any one of [1] to [5], wherein the dry gel is dried acrylamide.
[7] The first electrophoretic separation medium in the step (1) is a strip-shaped dried electrophoretic medium, and the first swelling solution is added to the dried first electrophoretic separation medium, and the dried The method according to any one of [1] to [6], wherein the first electrophoretic separation medium is swollen to a saturated state.
[8] The method according to any one of [1] to [7], wherein the first swelling solution contains an organic solvent having higher polarity than water.
[9] The dried gel has the following (a) and (b):
(a) consisting of sodium hydroxide and borate, glycine salt, citrate, phosphate, hydrogen phosphate, glutarate, acetate, phthalate, hydrogen phthalate and maleate At least one selected from the group;
(b) at least one selected from the group consisting of succinic acid, lactic acid, boric acid, citric acid, phosphoric acid, glutaric acid, acetic acid, phthalic acid and maleic acid, and borate, glycine salt, citrate, At least one selected from the group consisting of phosphate, hydrogen phosphate, glutarate, acetate, phthalate, hydrogen phthalate and maleate;
Comprising at least one group of components and negative ions,
The method according to any one of [1] to [8], wherein the gel obtained by swelling the dried gel in the step (4) has a pH of 6.8.
[10] The method according to any one of [1] to [9], wherein the second swelling solution in the step (4) is a buffer solution having a pH of 6.8 and further containing negative ions.
[11] In any one of [1] to [10], in the interval of the step (1), the first electrophoretic separation medium and the second electrophoretic separation medium are separated by a gas. The method described.
[12] The first electrophoretic separation medium in the form of a strip and the second electrophoretic separation medium are supported on one support substrate at an interval from each other,
A two-dimensional electrophoresis kit in which a dry gel that can swell and connect the first and second electrophoretic separation media is disposed at a position that does not contact the first electrophoretic separation media in the interval .
[13] The kit according to [12], wherein the support means further includes a second swelling solution supply means for swelling the dry gel after primary separation of the sample.

  Hereinafter, each step will be described.

Process (1)
In the two-dimensional electrophoresis substrate supplied in the step (1), the first electrophoresis separation medium is a gel that swells by absorption of water or an aqueous solution. The gel may be a dry gel or an already swollen gel, and preferably a dry gel is usually used.
An example of such a gel is a polyacrylamide gel. Such a gel is an IPG gel (immobilized pH gradient gel) that performs isoelectric focusing.

The shape of the first electrophoretic separation medium is not particularly limited, but is usually an elongated thin plate, for example, a strip. Both ends of the strip in the longitudinal direction may be provided with electrodes so that the first-dimensional electrophoresis can be performed.
The size of the first electrophoretic separation medium is not limited, but in the present invention, a very thin and thin shape can be employed even after swelling. For example, a material that has been swollen to a saturated state and that has a thickness of 0.1 to 1.0 mm and a length in the lateral direction of 0.5 to 5.0 mm can also be used. The length in the longitudinal direction varies depending on the size of the substrate and is not limited.

  A slab gel can be used as the second electrophoretic separation medium. A known slab gel can be used as the slab gel. The slab gel is generally an aqueous gel, and examples thereof include polyacrylamide gel, starch gel, and agar gel. The gel is preferably of a uniform concentration or concentration gradient.

  The first electrophoretic separation medium and the second electrophoretic separation medium are supported on one support means at a distance from each other. The second electrophoretic separation medium faces the first electrophoretic separation medium, and preferably the faces facing each other are parallel. In the present invention, during the swelling of the first electrophoretic separation medium and the first-dimensional electrophoresis, the first electrophoretic separation medium and the second electrophoretic separation medium are, for example, air in the interval. What is necessary is just to be separated by gas and does not need to partition with a liquid or solid.

  The interval differs depending on the size of two-dimensional electrophoresis and is not limited, but can be a relatively narrow gap of preferably 0.5 to 5 mm, more preferably about 1 to 3 mm.

Samples that can be used in the present invention include DNA, RNA, or protein. Of these, the present invention is particularly effective for protein separation. The protein is preferably a water-soluble protein.
Prior to electrophoresis, samples such as proteins can be fluorescently labeled. At this time, the isoelectric point marker can be electrophoresed under the same conditions.

  In this specification, “swelling” means that the gel (including dry gel) absorbs the solution to increase the volume, and “swelling solution” means a solution added when the gel is swollen. The “swelled gel” means a swollen gel.

<Dry gel>
The dried gel swells when the second swelling solution is added.
Such a dry gel is not limited as long as it has the property of absorbing the second swelling solution and swells as desired. For example, agarose, acrylamide, and the like can be used. Is preferred.

  A place where the dry gel is arranged in the interval is a position where the dry gel is not in contact with the first electrophoretic separation medium. This is to avoid adverse effects on the first-dimensional electrophoresis.

  A dry gel is preferably provided on the substrate. Further, it may be provided on the substrate in contact with the surface of the second electrophoretic separation medium facing the first electrophoretic separation medium. It is preferable that neither is in contact.

  The dry gel should be pre-designed to add a swelling solution to swell and thereby connect the first and second electrophoretic separation media.

Such a dry gel is obtained by drying a raw material gel. Such a raw material gel is preferably one that can regenerate a swollen gel having substantially the same dimensions as the raw material gel when it is dried and swollen again. By using such a raw material gel, the dried gel swells also in the direction of the facing surfaces of the first and second electrophoretic separation media, and the two media can be interconnected.
Therefore, it is preferable that the raw material gel is of such a size that the first and second electrophoretic separation media are connected and the interval is substantially sealed. Specifically, the raw material gel has a width and a height connecting the facing intervals of the first and second electrophoretic separation media.

That is, normally, the first electrophoretic separation medium and the second electrophoretic separation medium are preferably provided on the substrate so that the surfaces facing each other are parallel to each other, but the raw material gel has the following dimensions. It is preferable.
Raw material gel width: a width approximately equal to the average distance (referred to as “intermediate width” in this specification) between the surfaces of the first electrophoretic separation medium and the second electrophoretic separation medium facing each other. .
Raw material gel height: At least the height of the first electrophoretic separation medium from the substrate (referred to herein as the “primary medium height”).
Raw material gel length: At least approximately the same length as the length of the first electrophoretic separation medium in the longitudinal direction (referred to as “primary medium length” in this specification).

In the present specification, the “raw material gel width” refers to the distance between the two side surfaces of the raw material gel corresponding to the surface of the raw material gel facing the first electrophoretic separation medium and the second electrophoretic separation medium. Is the distance.
In this specification, “raw material gel height” is the size from the bottom surface to the top surface of the raw material gel when the raw material gel is placed on the substrate surface.
In this specification, the “raw material gel length” is the length in the longitudinal direction of the raw material gel corresponding to the longitudinal direction of the first electrophoretic separation medium.

When drying such a raw material gel, it is preferable to dry it while compressing in the direction opposite to the expansion direction in the raw material gel width direction.
Thus, when the raw material gel is compressed in advance to produce a dry gel, it can be rapidly swollen in the direction opposite to the compression direction during swelling. For this reason, when the dried gel compressed and dried is arranged in an appropriate direction, the first and second electrophoretic separation media can be interconnected very quickly due to swelling of the dried gel.
Further, by compressing in this way, the size can be reduced. In particular, when the width between the media is extremely small, compression in the raw material gel width direction is effective.

  The compression ratio depends on the type of dry gel used, but when compared with the case of drying without compression and the case of drying after compression, (compressed width) / (width without compression) ) × 100 (%) is preferably (30) to (80)%.

  The drying of the raw material gel may be carried out by exposure to air. The drying temperature is not limited as long as the raw material gel does not change in quality, and is, for example, about room temperature to 50 ° C.

Process (2)
A well-known thing can be used for the 1st swelling solution of the 1st electrophoresis separation medium, and it is not limited.
The first swelling solution is pure water, a water-soluble solution, or the like. The water-soluble solvent is not particularly limited as long as it does not adversely affect isoelectric focusing, and for example, an aqueous sodium chloride solution can be used.

  Furthermore, the first swelling solution preferably contains an organic solvent having a higher polarity than water. That is, the first swelling solution preferably contains water and an organic solvent having higher polarity than water.

Among organic solvents having a higher polarity than water, an organic acid having a higher polarity than water and an organic base having a higher polarity than water are preferable. Of these, organic acids are preferred. Among the organic acids, for example, formic acid, propionic acid, trifluoroacetic acid and the like are preferable, formic acid and trifluoroacetic acid are more preferable, and formic acid is particularly preferable.
The polarity of the solvent can be measured by a method such as dielectric constant measurement.

The content ratio of the organic solvent having a polarity higher than that of the water in the buffer solution (the volume of the organic solvent / the total volume of the aqueous solution × 100 (%)) is preferably 5% by volume or more, more preferably 10% by volume or more, and more. Preferably it is 15 volume% or more. The upper limit of the content is not particularly limited as long as it is in a range that does not adversely affect the first electrophoretic separation medium and the sample. For example, it is preferably 80% by volume or less, more preferably 60% by volume or less. More preferably, it is 30 volume% or less.
When the content of the organic solvent having a polarity higher than that of water is not less than the above amount, the swelling time of the swelling solution of the dried first electrophoretic separation medium can be significantly shortened. In particular, it is suitable for rapid swelling of IPG gel of polyacrylamide gel.

  The saturated state refers to a state in which the first swelling solution is swelled in the gel to the maximum extent, and even if the swelling solution is added, the content of the solution remains at a certain limit and does not increase any further.

  The first swelling solution further includes a protein denaturant such as urea or thiourea, a surfactant such as 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid, a reducing agent such as dithiothreitol, It may contain amphoteric compounds such as carrier ampholite (Biolite: manufactured by Biorat Laboratories).

  In the present invention, a sample to be subjected to two-dimensional electrophoresis can be mixed into the first swelling solution and impregnated in the first electrophoresis separation medium together with the first swelling solution.

Step (3)
After the first electrophoretic separation medium is swollen with the first swelling solution, current is passed from both ends in the longitudinal direction of the swollen first electrophoretic separation medium to perform the first-dimensional separation. The first swelling solution functions as a first-dimensional migration medium as it is.

The temperature of the first electrophoretic separation medium may be controlled to a temperature that does not adversely affect the first-dimensional gel or sample. For example, it is preferably 0 ° C. to 20 ° C. while the electric field is supplied. .
The temperature control method is not particularly limited. For example, the Peltier element can be provided on the back side of the first electrophoretic separation medium or on the back side of the support means to which the first electrophoretic separation medium is coupled.

The power supply method is preferably such that the amount of power per unit volume is from 1 to 120 mW / mm ³ , more preferably from 5 to 100 mW / mm ³ , more preferably from 10 to 60 mW / mm, immediately after the start of supply of the electric field. Supply electric field in the range of ³ . As described above, when a large amount of electric power of a certain amount or more is supplied immediately after the start of the first-dimensional electrophoresis, the first-dimensional separation time can be significantly shortened.

  By performing the first-dimensional separation as described above, the separation time can be shortened, so that the solution impregnated in the first electrophoretic separation medium evaporates and the first electrophoretic separation medium deteriorates. Can be suppressed. That is, conventionally, in order to prevent evaporation of the solution, the first-dimensional gel was protected with paraffin oil or a non-conductive solid during the first-dimensional electrophoresis (for example, Patent Documents 3 and 4). However, according to the power supply method as described above, it is not necessary to provide such a liquid or solid, and the first-dimensional electrophoresis step becomes extremely simple.

Process (4)
After performing the primary separation, a second swelling solution is added to the dried gel, and the dried gel is swollen until the distance between the first electrophoresis separation medium and the second electrophoresis separation medium is connected. Let Thereby, the first electrophoretic separation medium and the second electrophoretic separation medium are interconnected by the swollen gel obtained by swelling the dry gel.

  The swollen gel obtained by swelling the dried gel is preferably in contact with at least the entire surface of the first electrophoretic separation medium facing the second electrophoretic separation medium.

  The second swelling solution is preferably an aqueous solution such as a buffer solution or water.

  Buffers generally include, for example, tris-borate buffer, tris-hydrochloric acid buffer, tris-tricine buffer, tris-sodium dihydrogen phosphate buffer, borate buffer, phosphate buffer, etc. Examples include buffers used as electrophoresis buffers for proteins and the like. The buffer may be used at a concentration generally used as a protein buffer.

  The pH of the second swelling solution is preferably 6-8. Adjustment of the pH of the liquid buffer is sodium hydroxide, hydrochloric acid, boric acid, glycine-hydrochloric acid buffer, citric acid-disodium phosphate buffer, β-β′dimethylglutaric acid-sodium hydroxide buffer, acetic acid- A sodium acetate buffer, potassium hydrogen phthalate-sodium hydroxide buffer, sodium maleate-sodium hydroxide buffer, phosphate buffer or the like can be used.

  More preferably, the second swelling solution has a pH of 6.8 and can contain negative ions. Examples of the negative ions include halogen atom ions such as chlorine ions, fluorine ions, bromine ions, and iodine ions. Of these, chloride ions are preferred. The negative ion concentration is preferably 0.1 to 0.2M.

Preferably, the dry gel has the following (a) and (b):
(a) consisting of sodium hydroxide and borate, glycine salt, citrate, phosphate, hydrogen phosphate, glutarate, acetate, phthalate, hydrogen phthalate and maleate At least one selected from the group;
(b) at least one selected from the group consisting of succinic acid, lactic acid, boric acid, citric acid, phosphoric acid, glutaric acid, acetic acid, phthalic acid and maleic acid, and borate, glycine salt, citrate, At least one selected from the group consisting of phosphate, hydrogen phosphate, glutarate, acetate, phthalate, hydrogen phthalate and maleate;
Comprising at least one group of components and negative ions,
The gel obtained by swelling the dried gel has a pH of 6.8.
The gel obtained by swelling the dried gel in the step (4) includes the one group of components and negative ions.

  After the first-dimensional electrophoresis, the sample is separated in the longitudinal direction of the first electrophoretic separation medium, but may be separated as a broad spot in the second-dimensional electrophoresis direction. When the second gel is swollen using the second swelling solution under the above conditions to connect the first electrophoretic separation medium and the second electrophoretic separation medium, and the second-dimensional electrophoresis is performed, When the sample passes through the swollen gel, the sample existing in the broad can be concentrated in a narrow range in the electrophoresis direction of the second dimension in the gel. As a result, the accuracy of second-dimensional electrophoresis can be improved.

  The second swelling solution may further contain a suitable surfactant, such as sodium dodecyl sulfate (SDS).

Process (5)
The individual components developed in the first-dimensional gel move through the swollen swollen gel and move from the first-dimensional gel to the nearest second-dimensional gel, and are developed according to the molecular weight. At this time, the molecular weight marker is usually electrophoresed under the same conditions.
The second-dimensional electrophoresis may be performed in the same manner as a known method, and is not particularly limited. Second dimension electrophoresis can be performed by, for example, tris-glycine buffer, tris-borate buffer, tris-hydrochloric acid buffer, tris-tricine buffer, tris-sodium dihydrogen phosphate buffer, borate buffer, It can carry out using the buffer solution generally used as a buffer solution for electrophoresis, such as protein, such as a phosphate buffer solution. The buffer may be used at a concentration generally used as a protein buffer.

  The buffer may further contain a suitable surfactant: for example, sodium dodecyl sulfate (SDS), glycine and the like.

  The pH of the buffer is preferably 6-10. The pH of the buffer solution is adjusted by sodium hydroxide, hydrochloric acid, boric acid, glycine-hydrochloric acid buffer solution, citric acid-disodium phosphate buffer solution, β-β′dimethylglutaric acid-sodium hydroxide buffer solution, acetic acid-acetic acid A sodium buffer, potassium hydrogen phthalate-sodium hydroxide buffer, sodium maleate-sodium hydroxide buffer, phosphate buffer or the like can be used.

  The second dimensional migration is usually performed preferably at 1 to 10 V / mm for about 5 to 20 minutes.

  A spot of each component such as protein is confirmed on the gel after the second dimensional migration. Examples of methods for detecting proteins subjected to electrophoresis include absorption by light and fluorescence detection, electrochemical detection, chemistry and biochemiluminescence detection, mass detection, and thermal detection.

  For example, if a protein is fluorescently labeled, individual spots can be confirmed by tracking the fluorescence. In addition, protein spots can also be confirmed by CBB staining or silver staining. Each component such as protein thus separated can be identified based on the coordinate information by comparing with the result of two-dimensional electrophoresis performed under the same conditions. Alternatively, in order to proceed with the analysis of the unknown protein, it is possible to further identify the protein at each spot.

<Two-dimensional electrophoresis kit and device>
In the two-dimensional electrophoresis kit according to the present invention, the first electrophoretic separation medium in the form of a strip and the second electrophoretic separation medium are carried on one support substrate at a distance from each other,
A dry gel that can swell and connect the first and second electrophoretic separation media is disposed at a position that does not contact the first electrophoretic separation medium in the interval.

  The support means may further include a second swelling solution supply means for swelling the dry gel after primary separation of the sample.

  Further, a first swelling solution supply means may be provided for swelling the dried first electrophoretic separation medium.

  Such a two-dimensional electrophoresis kit has a low-cost and simple structure so that it can be disposable to prevent contamination of the sample.

The first electrophoretic separation medium, the second electrophoretic separation medium, the first swelling solution, and the second swelling solution are as described above. The supporting means may be any means that supports the first electrophoretic separation medium and the second electrophoretic separation medium, such as a plastic substrate, a glass substrate, a metal substrate whose surface is electrically insulated, and a surface. And a semiconductor substrate in which is electrically insulated.
The second swelling solution supply means is provided in the supporting means, whereby the swelling solution can be easily added to the dried gel.

  The first swelling solution supply means is provided in the supporting means, whereby the first swelling solution can be easily added to the dried first electrophoretic separation medium.

  The two-dimensional electrophoresis apparatus according to the present invention includes an electrode and the two-dimensional electrophoresis kit.

  The present invention will be further described below with reference to the drawings, but the present invention is not limited to these.

FIG. 1 is an example of a cross-sectional view of a two-dimensional electrophoresis kit according to the present invention.
The first electrophoretic separation medium 1 and the second electrophoretic separation medium 2 are provided with a gap (junction portion) 7 on a support substrate 6. The first electrophoretic separation medium 1 and the second electrophoretic separation medium 2 are arranged to face each other in parallel with each other. Further, second-dimensional electrophoresis buffer tanks 3 and 4 are provided. In the interval 7, the dry gel 5 is provided on the substrate 6.

  The first electrophoretic separation medium 1 is dried before use, but the first swelling solution is added immediately before use. The first swelling solution is preferably added in such an amount that it does not flow down from the first electrophoretic separation medium 1 due to surface tension. After the swelling, as in step (3) of the above-described method, the first-dimensional electrophoresis is performed by supplying power from both ends in the longitudinal direction of the first electrophoretic separation medium 1.

After the first-dimensional electrophoresis, the second swelling solution is added to the dried gel 5 to swell the dried gel 5.
FIG. 2a shows a cross-sectional view and a top view of a two-dimensional electrophoresis kit in which a dry gel is arranged, and FIG. 2b shows a cross-sectional view and a top view of the state after the dry gel is swollen to interconnect the intervals. .
The dried gel 5 in FIG. 2a is obtained by drying a raw material gel having the same size as the interval 7 so as to swell in the X direction and the Y direction. By adding a second swelling solution, the dried gel 5 is dried as shown in FIG. 5 swells, and the first electrophoretic separation medium 1 and the second electrophoretic separation medium 2 can be connected by the swelling gel 8. Here, when the raw material gel is compressed and dried, the swelling ratio can be improved and the size of the dried gel 5 can be reduced. In particular, when compressed in the Y direction and dried, the size in the Y direction can be reduced, and the swelling ratio in the Y direction can be increased and improved.

  Next, the second-dimensional electrophoresis can be performed by passing the electrodes through the buffer solution tanks 3 and 4.

Further, a Peltier element can be provided on the back side of the first electrophoretic separation medium 1 or on the back side of the support means 1 at the position where the first electrophoretic separation medium 1 is coupled (not shown).
Thereby, in the first-dimensional electrophoresis, the first electrophoresis separation medium 1 can be simply and efficiently cooled with energization.

As shown in FIG. 3, the second swelling solution can also be performed using a swelling solution supply means 9 provided in the supporting means 1. The swelling solution supply means 9 is a liquid reservoir for releasing the second swelling solution.
The support means 6 may be provided with a first swelling solution supply means for swelling the first electrophoretic separation medium 1 in a dry state (not shown). The means for supplying the swelling solution is a reservoir for releasing the first swelling solution.
When the second swelling solution supply means 9 and the first swelling solution supply means are provided, the operation can be automated, and rapid and accurate electrophoresis can be performed. In particular, for example, even when the width of the dry gel 5 is several millimeters or the interval 7 is several millimeters, there is an advantage that these solutions can be supplied accurately.

  According to the present invention, the one-dimensional gel and the two-dimensional gel can be rapidly and simply prepared by preliminarily providing the dry gel in the interval, and pouring the swelling solution after the primary separation and before the secondary separation. It can be easily interconnected. Further, the total two-dimensional electrophoresis can be performed in a short time by an extremely simple operation, for example, the swelling time can be further shortened by using a specific swelling solution for swelling the first electrophoretic separation medium.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
[Example 1]
The connection method between the first-dimensional gel and the second-dimensional gel after the first-dimensional electrophoresis was tested.

  As shown in FIGS. 1, 2, and 3, a polyacrylamide dry pH gradient immobilized gel (Invitrogen, thickness 0.02 mm × width in the short direction) is used as the first-dimensional electrophoresis gel 1 (gel 1). 0.8 mm × length 52 mm in the longitudinal direction) was placed at a predetermined position on the plastic support substrate 6.

Second-dimensional electrophoresis gel 2 (gel 2) was a polyacrylamide gel, and 375 mM Tris-HCl buffer (pH 8.8) was used as the buffer solution.
The side of the longitudinal direction of the gel 1 is blocked by a space so as not to contact the wall of the support substrate 6, and is arranged so that the gel 1 and the gel 2 do not contact each other, and the gel 1 and the gel 2 are electrically connected to each other. Is insulated.

  A polyacrylamide gel having a size of the gap 7 between the gel 1 and the gel 2 was prepared. 125 mM Tris-HCl buffer (pH 6.8) was used as the buffer. The gel was dried by fixing the top and bottom in the longitudinal direction, and the width and thickness in the lateral direction were shrunk. This dried gel was placed in the gap 7 of the support substrate 6 as the gel 5 shown in FIGS.

Distilled water was poured into the gel 5 from a liquid reservoir provided on the substrate. After 20 minutes, distilled water was added again. The gel 5 expanded, and after 30 minutes, the gel 1 and the gel 2 were connected by expanding until the gap 7 was eliminated. FIG. 2 shows a schematic diagram in which the gel 5 expands to become a gel 8 in which the gel 1 and the gel 2 are connected.
FIG. 4 shows a photograph in which gel 5 expands and gel 1 and gel 2 are connected.
[Example 2]
Two-dimensional electrophoresis was performed using the connection method shown in Example 1.

  The first-dimensional gel (1), the second-dimensional gel (2), and the dried gel (5) were placed on the support substrate 6 in the same manner as shown in Example 1.

  Next, protein denaturant (urea 6M, thiourea 2M), surfactant (CHAPS (manufactured by Dojindo)) 2% (w / v), reducing agent (dithiothreitol) 20 mM, carrier ampholite (manufactured by Invitrogen) A swelling solution using water containing 0.5% (v / v) as a solvent was prepared, a sample sample was added to the swelling solution, and 20 μL of a sample-containing swelling solution was prepared. As a sample, a marker protein for two-dimensional electrophoresis (amyloglucosidase, ovalbumin, carbonic anhydrase, myoglobin) (manufactured by Sigma) was used.

  The swelling solution containing the sample was added to the gel 1 using a liquid reservoir attached to the substrate, and the gel 1 was swollen. The addition was allowed to stand for 15 minutes, and then the isoelectric focusing was performed by bringing the first-dimensional electrophoresis electrode 10 into contact with the longitudinal end of the gel 1.

Apply electric power within 10mW / mm ³ between electrodes (0-5000 V (linear) for 4 minutes, 5000-6000 V (linear) for 1 minute, 6000 V for 5 minutes), and apply electricity for a total of 10 minutes. Washed away. At this time, in order to prevent drying and burning of the gel due to the generation of heat, the gel 1 was cooled by a Peltier device so that the temperature became 0 to 5 ° C.

  Distilled water was injected into the gel 5 in the same manner as in Example 1 to expand the gel 5 and connect the gel 1 and the gel 2 together.

Next, a buffer solution for second-dimensional electrophoresis (containing 0.19 M glycine, SDS, and 25 mM trisaminomethane) is injected into the buffer solution chambers 3 and 4, and a buffer solution chamber close to the first-dimensional gel as a cathode. 3, the electrode 11 was brought into contact with the buffer solution tank 4 close to the second-dimensional gel as an anode, and the second-dimensional electrophoresis was performed (constant voltage 200 V, about 15 minutes).
FIG. 5 shows an image of a second-dimensional polyacrylamide gel in which the sample is separated after the second-dimensional electrophoresis performed following the first-dimensional electrophoresis.

FIG. 1 is an example of a cross-sectional view of a two-dimensional electrophoresis kit according to the present invention. 2a and 2b are examples of a cross-sectional view and a top view of a usage mode of the two-dimensional electrophoresis kit according to the present invention. FIG. 2a is a cross-sectional view and a top view of a two-dimensional electrophoresis kit in which a dry gel is arranged, and FIG. . FIG. 3 is an example of a schematic diagram of a two-dimensional electrophoresis kit having a second swelling solution injection device. FIG. 4a is a photograph from the upper surface of the two-dimensional electrophoresis kit in a state where a dry gel is arranged, and FIG. It is the photograph from the upper surface of the kit for two-dimensional electrophoresis in the state which connected the electrophoresis separation medium (2D). FIG. 5 is a photograph showing the result of separation of fluorescently labeled proteins (amyloglucosidase, ovalbumin, carbonic anhydrase, myoglobin) in the second electrophoretic separation medium by second-dimensional electrophoresis. .

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st electrophoresis separation medium 2 2nd electrophoresis separation medium 3 Buffer solution tank 4 Buffer solution tank 5 Dry gel 6 Support substrate 7 Space | interval (junction)
8 Swelling gel 9 Second swelling solution supply means 10 Electrode 11 Electrode

Claims (13)

A two-dimensional electrophoresis method comprising the following steps:
(1) A first electrophoretic separation medium in the form of a strip and a second electrophoretic separation medium are supported on a single support substrate at an interval from each other, and swell to form the first and second electrophoretic separation media. Providing a two-dimensional electrophoretic substrate, wherein a dry gel capable of connecting an electrophoretic separation medium is disposed at a position not in contact with the first electrophoretic separation medium during the interval;
(2) impregnating the first electrophoretic separation medium with a sample;
(3) applying an electric field to the first electrophoretic separation medium to primarily separate components in the sample;
(4) After the primary separation, a second swelling solution is added to the dried gel, and the first electrophoretic separation medium and the second electrophoretic separation medium are connected by the swollen gel. Swell the dried gel, and (5) both the first electrophoretic separation medium and the second electrophoretic separation medium are substantially in the longitudinal direction of the first electrophoretic separation medium. Applying an electric field in a vertical direction to the second electrophoretic separation medium to secondary-separate the primary separated components.   The method of claim 1, wherein the dry gel is disposed on the spaced substrate. The dried gel is
In the step (1), the first electrophoretic separation medium and the second electrophoretic separation medium are disposed at positions that do not come into contact with each other,
The method according to claim 1 or 2, wherein in the step (4), the gel swells in the direction of the first electrophoretic separation medium and the direction of the second electrophoretic separation medium.   The dry gel is a gel contracted by drying a raw material gel, and the raw material gel has a size capable of connecting between the first electrophoretic separation medium and the second electrophoretic separation medium. The method according to any one of claims 1 to 3.   The method according to claim 4, wherein the dry gel is obtained by drying the raw material gel while being compressed.   The method according to claim 1, wherein the dried gel is dried acrylamide.   The first electrophoretic separation medium in the step (1) is a strip-shaped dry electrophoretic medium, and a first swelling solution is added to the dried first electrophoretic separation medium, and the dried first electrophoretic medium is added. The method according to claim 1, wherein the electrophoretic separation medium is swollen to a saturated state.   The method according to claim 1, wherein the first swelling solution contains an organic solvent having a higher polarity than water. The dried gel has the following (a) and (b):
(a) consisting of sodium hydroxide and borate, glycine salt, citrate, phosphate, hydrogen phosphate, glutarate, acetate, phthalate, hydrogen phthalate and maleate At least one selected from the group;
(b) at least one selected from the group consisting of succinic acid, lactic acid, boric acid, citric acid, phosphoric acid, glutaric acid, acetic acid, phthalic acid and maleic acid, and borate, glycine salt, citrate, At least one selected from the group consisting of phosphate, hydrogen phosphate, glutarate, acetate, phthalate, hydrogen phthalate and maleate;
Comprising at least one group of components and negative ions,
The method according to any one of claims 1 to 8, wherein the gel obtained by swelling the dried gel in the step (4) has a pH of 6.8.   The method according to any one of claims 1 to 9, wherein the second swelling solution in the step (4) is a buffer solution having a pH of 6.8 and further containing negative ions.   The method according to claim 1, wherein the first electrophoretic separation medium and the second electrophoretic separation medium are separated by a gas in the interval of the step (1). The strip-shaped first electrophoretic separation medium and the second electrophoretic separation medium are carried on one support substrate at an interval from each other,
A two-dimensional electrophoresis kit in which a dry gel that can swell and connect the first and second electrophoretic separation media is disposed at a position that does not contact the first electrophoretic separation media in the interval .   The kit according to claim 12, wherein the supporting means further comprises a second swelling solution supply means for swelling the dried gel after primary separation of the sample.