JPH0718840B2 - Two-dimensional electrophoresis method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (A) Field of Industrial Application The present invention relates to a two-dimensional electrophoresis method.

(B) Conventional technology In the conventional two-dimensional electrophoresis, after completion of the first-dimensional electrophoresis, the first-dimensional gel is extruded from the glass tube to be in a free state and then moved to move the second-dimensional gel. It was bonded to the migration start side.

(C) Problems to be solved by the invention However, it takes a great deal of skill to take out the first-dimensional gel from the tube and bond it to the second-dimensional gel in a normal state. The gel may break, stretch, or bend during the operation.

The present invention has been made in view of the above points, and a main object thereof is to provide a two-dimensional electrophoresis method capable of performing the next two-dimensional electrophoresis without taking out the first-dimensional gel. .

(D) Means for Solving the Problems The present invention is that the first-dimensional gel and the second-dimensional gel are vertically adjacent to each other, and a liquid insulating band and a conductive band are appropriately interposed between them, The first and second dimensions of electrophoresis are performed in a state where both gels are held in a fixed position relative to each other. The more detailed structure is as follows. The gel for electrophoresis is vertically adjacent to each other via a liquid insulating band, the first-dimensional electrophoresis is performed in the first-dimensional gel for electrophoresis, and the liquid insulating band is removed after the completion of the first-dimensional electrophoresis. The two-dimensional electrophoresis method is characterized in that the two gels are substantially and electrically connected to each other by substituting the electroconductive band made of the electrophoretic material, and second-dimensional electrophoresis is performed in that state.

(E) Action The presence of a liquid insulating band causes first-dimensional electrophoresis in a state in which both gels are held vertically and in an insulating state, and the presence of a conductive band substantially connects both gels electrically. Electrophoresis of the second dimension is performed in the state where it is subjected.

(F) Embodiments The present invention will be described in detail below with reference to the drawings, but the present invention is not limited to the following embodiments.

First, FIG. 1 shows the disassembled constitution of the gel cassette (X) used in the present invention, and FIG. 2 shows its front constitution.
(1) is a substrate, and (2) is a surface plate, which is a rectangular shape having substantially the same shape. The substrate (1) is made of a heat conductive material and has a so-called cooling function of releasing heat generated during electrophoresis. As the surface plate (2), a transparent or opaque plastic plate is appropriately used, and the surface plate (2) and the substrate (1) are laminated via a spacer (3). It is fixed by a fixture. The spacer (3) is also made of plastic and comprises both side parts (4) and (4) and a lower part (6) having an inclined recess (5), the front and rear surfaces of which are between the substrate (1) and the surface plate (2). And is in contact with both side portions and the lower position. (7) is a silicone rubber which is a sealing material for enhancing the adhesion between the substrate (1), the surface plate (2) and the spacer (3).

Since the spacer (3) is interposed between the substrate (1) and the surface plate (2) as described above, a gap space (8) having a constant thickness is provided between the substrate (1) and the surface plate (2). ) Is formed.

The lower end of the clearance space (8) of the surface plate (2), that is, the position corresponding to the lowest part of the inclined recess (5), and both sides slightly below the opening (9) at the upper end of the clearance space (8). Through holes (10), (11) and (11) are provided at positions facing each other.

The two-dimensional electrophoresis method of the present invention using the gel cassette (X) will be described below.

First, a gel solution for second dimension electrophoresis is injected into the interstitial space (8) through the through hole (10), for example, by driving a pump, and the two through holes (11) (11) in the interstitial space (8) are injected. A second-dimensional gel for electrophoresis is formed on a portion located below the position (H) connecting the two. As the gel for the second dimension electrophoresis, for example, a polyacrylamide concentration gradient gel is used, and the gel has T (weight percent) = 15% to
5%, C (weight ratio) = 4%, length x width x 160 mm x 1
It is a flat plate with a thickness of 60 mm and a thickness of 0.5 mm. The gel solution should be injected at the position (H) before the liquid surface reaches the position (H).
It stops 10 mm to 20 mm below that, and thereafter, for example, a 60% glycerin solution having a larger specific gravity than the gel solution is injected to further raise the liquid level and reach the position (H). In this state, the glycerin solution is located in the interstitial space (8) corresponding to the inclined recess (5).

After injecting the gel solution for the second dimension electrophoresis, or after the solution solidifies and gels, an insulating liquid that becomes a liquid insulating band through the two through holes (11) (11). To a height of 1 mm. As the insulating liquid, carbon tetrachloride or polyhalogen carbon liquid is used.

On the insulating liquid layer, a gel solution for the first-dimensional electrophoresis is further injected through the upper opening (9). A gel for polyacrylamide concentration gradient was also used as the gel for the first-dimensional electrophoresis, and the gel had T = 5.5% and C =
4%, Ampholine (LKB) 4%, size 5 × 160m
It is a flat plate with m and a thickness of 0.5 mm.

As described above, as shown in FIG. 3, the first-dimensional electrophoresis gel (13) and the second-dimensional electrophoresis gel (14) are vertically sandwiched by the insulating liquid (12) layer of about 1 mm. ) Is arranged in the gel cassette (X). Thus, since the insulating liquid (12) layer is provided, both gels (13) and (14) are not electrically affected by each other.

In this state, a voltage is applied between the electrodes at the positions A and B of the gel body, so that the first-dimensional electrophoresis is performed on the sample placed at the predetermined position of the first-dimensional gel for electrophoresis.

After completion of the migration, the insulating liquid (12) is drained through the through holes (11) (11), and the melted agarose is injected into the linear space remaining therein to solidify to form a conductive band. Since agarose is conductive and allows proteins to pass through, in this state, the first-dimensional electrophoresis gel (13) and the second-dimensional electrophoresis gel (14) are substantially and electrically connected. Becomes Then, a voltage is applied between the electrodes at the positions indicated by C and D, and second-dimensional electrophoresis is performed. After the completion of the second-dimensional electrophoresis, the surface plate (2) is removed and the gel is stained to discriminate the analysis result.

For example, Tris-aminomethane buffer (pH 8.6) may be used in place of the above agarose, but the thickness of the liquid layer is 3 mm or less, preferably 1 mm.

In addition, as a matter of course, the electrodes are appropriately formed on the portion of the gel cassette (X) facing the predetermined position of the gel.

(G) Effect of the Invention The two-dimensional electrophoresis method according to the present invention is configured as described above, and is bonded to the second-dimensional gel for electrophoresis without taking out the first-dimensional gel for electrophoresis. As a result, unlike the conventional case, there is no fear of damaging the gel for the first-dimensional electrophoresis, and the operability is extremely excellent. Further, in the two-dimensional electrophoresis method according to the present invention, the first-dimensional electrophoresis gel and the second-dimensional electrophoresis gel are vertically adjacent to each other through the liquid insulating band. It is not necessary to preliminarily solidify or gelate both gel solutions at a predetermined interval when arranging them inside a plate-shaped member or a gel cassette. That is, a second-dimensional electrophoresis gel is introduced into a plate-like member in a liquid state, and after solidification or gelation thereof, a predetermined amount of an insulating liquid to be an insulating band is introduced, and then the first-dimensional electrophoresis is performed. By introducing the gel for use in a liquid state and waiting for its solidification or gelation, both gels can be arranged at a predetermined interval from each other. Therefore, not only is no special device or member or skill required to solidify or gel the gel solutions at a predetermined interval from each other, but the time required for electrophoresis can be shortened.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of an embodiment of a gel cassette used in the present invention, FIG. 2 is a front view of the embodiment, and FIG. 3 is a front view of an embodiment of a gel body formed in the gel cassette. (X) …… Gel cassette, (1) …… Substrate, (2) ……
Surface plate, (3) …… Spacer, (8) …… Gap space,
(10) (11) (11) …… Through hole.

Claims (1)

[Claims] 1. A first-dimensional electrophoretic gel and a second-dimensional electrophoretic gel are vertically adjacent to each other through a liquid insulating band,
First-dimensional electrophoresis is performed in the first-dimensional electrophoresis gel, and after completion of the first-dimensional electrophoresis, the liquid insulating band is replaced with a conductive band made of a electrophoretic material so that both gels are substantially A two-dimensional electrophoresis method, which comprises electrically connecting and performing second-dimensional electrophoresis in that state.