JPH0641934B2 - Two-dimensional electrophoresis - Google Patents

Description

TECHNICAL FIELD The present invention relates to a two-dimensional electrophoresis method, and more particularly to a two-dimensional electrophoresis method including an improved one-dimensional gel making step.

Conventional technology Conventionally, a two-dimensional electrophoresis method in which a sample is electrophoresed in a one-dimensional gel, the resulting one-dimensional sample gel column is placed on the upper end of the two-dimensional gel, and further electrophoresed. In, the sample solution is added to a cylindrical one-dimensional gel, and the components are separated (one-dimensional separation) by the migration,
The cylindrical gel fixed in that state was placed on the upper end of the two-dimensional gel to perform two-dimensional migration.

Problems to be Solved by the Invention In this case, since the cylindrical one-dimensional separated gel is placed on the upper end of the two-dimensional gel, a gap is formed on both sides of the contact surface of both gels,
This was one of the causes of the migration disorder.

Means for Solving Problems In order to solve such problems, the present invention provides a one-dimensional gel obtained by arranging a plurality of parallel rod-shaped gel chambers having a rectangular cross section in a two-dimensional electrophoresis method. A one-dimensional gel is prepared in at least one of the rod-shaped gel chambers in the preparation device, and a one-dimensional separated gel of a sample having a rectangular cross section is prepared by subjecting the sample to electrophoresis in the rod-shaped one-dimensional gel, By laying the original separated gel on the upper end of the two-dimensional gel, the lower surface of the one-dimensional separated gel and the upper end surface of the two-dimensional gel are electrophoresed in close contact with each other without gaps. This is an electrophoretic method.

Action The present invention, in the above-mentioned constitution, is one in which the one-dimensional separated gel is brought into close contact with the two-dimensional gel without a gap, and this adhesiveness causes a gap on both sides of the connection surface, which causes migration. It completely eliminates the drawback of the conventional cylindrical one-dimensional gel that is disturbed.

According to a preferred development of the invention, the zero-dimensional migration is performed as a pre-step of the one-dimensional migration so that, for example, when analyzing a protein sample, the protein molecules in the sample individually encounter free radicals. The sample is concentrated so as to avoid the above, which enables effective execution of one-dimensional migration and further two-dimensional migration.

Therefore, the examples described below are applied to the two-dimensional migration method including "zero-dimensional migration" as the sample pretreatment.

Description of Examples Zero-dimensional migration Zero-dimensional migration as referred to here is a process of concentrating and forming a sample gel inserted into a one-dimensional gel by electrophoresis instead of injecting a sample solution into the one-dimensional gel. That is. In this zero-dimensional electrophoresis, a gel container GC ₀ having one or two or more rows of prismatic gel chambers (1) as shown in FIG. 1 (a) is used as an upper buffer tank (2) and a lower buffer tank (3). ), The gel chamber (1) contains, for example, a polyacrylamide gel as an electrophoretic medium for a protein sample, and the sample solution is introduced from above.
In this case, an appropriate voltage is applied between the anode A provided in the upper buffer tank (2) and the cathode B provided in the lower buffer tank (3). Before this migration, 2-mercaptoethylamine as a radical scavenger is pre-migrated on the polyacrylamide gel, whereby the consumption of the sample protein can be prevented. Further, during migration of the sample solution, cysteine as a reducing agent is also co-migrated, thereby preventing SS cross-linking of the protein molecule. The sample thus electrophoretically concentrated is taken out from the gel container GC ₀ or taken out by disassembling the container, and then the appropriate portion of the concentrated gel portion (4) is cut out to prepare a sample gel piece SG for one-dimensional gel. And

The upper buffer tank used for this zero-dimensional migration
(2) and the lower buffer tank (3) have the same structure as the upper buffer tank and the lower buffer tank for one-dimensional electrophoresis described below, respectively. The only difference in the mechanism is that a gel container having a shorter total length than the above is used. The distance between both plates of the gel container GC ₀ , that is, the thickness of the gel is about 3 mm in this case, but it can be made from about 1 mm to more than 3 mm according to its use. The same applies to the gel thickness for one-dimensional migration and two-dimensional migration.

One-dimensional migration Gel container GC ₁ for one-dimensional migration is a prismatic column as shown in Fig. 2 (a), that is, a plurality of rows of gel chambers having a rectangular cross section.
In addition to having (10), a sample insertion window (11) communicating with each gel chamber (10) is formed at a relatively high level position on one side surface. The sample gel piece by the zero-dimensional treatment has a height slightly lower than the vertical dimension of this window, but since the zero-dimensional and this one-dimensional gel chamber width and depth are the same, the gel chamber from this window (10) Each of the sample gel pieces SG inserted in the section enters the excision portion corresponding to this window in the electrophoretic polyacrylamide gel in the same room, and adheres well to the entire upper and lower cut ends. SGC shown in FIG. 2 (b) is a sample gel cover plate, wherein each window of the gel container GC ₁
It has a plurality of window stoppers (12) corresponding to (11). Each window plug (12) is inserted into each corresponding window (11), thereby supporting and covering the side surface of the sample gel piece SG at the corresponding position in the gel chamber (10). The gel container GC ₁ equipped with the sample gel cover SGC is the third
As shown in the figure, the upper and lower ends are attached to the upper buffer tank ABV ₁ and the lower buffer tank CBV ₁ , and an appropriate voltage is applied between the anode A and the cathode C to perform the electrophoretic separation of the sample gel. Is. The sample gel cover SGC is attached to the gel container GC ₁ by using a clip (13) as shown in FIG. 4, for example. Also, the upper buffer tank ABV ₁ and the lower buffer tank C
The internal structure of BV ₁ is also as shown in FIG.

The polyacrylamide gel in the above one-dimensional migration is
Before inserting the sample gel piece SG, 2-mercaptopropionic acid as a radical scavenger is pre-migrated, and further, during migration of the sample gel, the same 2-mercaptopropionic acid as a radical scavenger is used as a reducing agent. Are simultaneously electrophoresed to maintain a strong reducing state in the gel.

As described above, the method of preparing the sample gel piece separately from the one-dimensional gel and inserting it into the one-dimensional gel is a unique method adopted only in the present invention, whereby the pre-electrophoresis treatment of the radical scavenger is performed. Together with this, the problem of sample consumption due to residual radicals has been solved.

Two-dimensional migration A two-dimensional migration gel container is performed by combining three types of gel container plates LP, MP, and RP as shown in FIG. 5, and connecting the gel container structure thus formed to the upper and lower buffer tanks. . When two or more two-dimensional gel chambers are used, the space between the MP-MP plate surfaces and the left container plate LP
Is formed between the right side surface of the container plate MP and the left side surface of the corresponding container plate MP, and between the left side surface of the right side container plate RP and the right side surface of the corresponding container plate MP.

The cross section of the structure in which the container structure GC ₂ is combined with the upper buffer tank ABV ₂ and the lower buffer tank CBV ₂ is as shown in FIG. 6, and each gel chamber has an allowance for mounting the one-dimensional migrated gel on the upper end. The gel BG for two-dimensional electrophoresis is inserted while leaving the above. The slot D ₁ is formed at the upper end of the container plate MP, and the upper end outer surfaces of the left container plate LP and the right container plate RP and the upper buffer tank AB are formed.
A similar slot D ₂ is formed between the protrusion formed at the lower end of V ₂ . The one-dimensional migrated gel SG ₂ is inserted from the upper end of each gel chamber of the container structure GC ₂ , and
Gauze is applied to the upper end of the container structure GC ₂ , and the flat wedge PW is inserted into the slots D ₁ and D ₂ from above the gauze G, and the gauze tensioned by this inserts the one-dimensional migrated gel SG _{2 into} the gel. The upper edge is pressed so that it is in close contact with the upper end of the two-dimensional electrophoresis gel BG.

FIG. 7 shows the internal structure of the fastening member H for maintaining the container structure GC ₂ , the upper buffer tank ABV ₂ and the lower buffer tank CBV ₂ .

Also in this two-dimensional migration, residual free radicals are removed by migrating 2-mercaptoethylamine as a radical scavenger on the polyacrylamide gel, which is the migration medium, before migration of the sample gel column SG ₂ component, and During migration of the column component of the sample gel, cysteine was co-migrated together as a reducing agent having a charge to maintain a strong reducing state in the two-dimensional gel. at the same time,
By removing the basic radical promoter in the gel by pre-electrophoresis and adding hydrochloric acid to the lower part, that is, the negative electrode buffer tank CBV ₂ , the pH of the gel was lowered from normal 4.5 to about 0.9. To be 3.6.

FIG. 8 is a graph showing the relationship between the molecular weight and the migration distance of the sample protein obtained by carrying out the method of the present invention including “zero-dimensional migration”. In this case, if the difference in the effect of charge on the mobility is eliminated, the logarithmic value of the molecular weight is almost completely proportional to the mobility, indicating that the molecular weight can be measured.

As is clear from the separation model of the two-dimensional gel shown in the graph of FIG. 8 and FIG. 9, from the basic region of the two-dimensional gel, four spots (A, B, C, D) were detected. In FIG. 9, spot A is found at the lower right of L33, spot B is at the lower left of L34, spot C is midway between L29 and L27, and spot D is found just below L32. In addition, A,
B and C are present in the 50S subparticle and D is present in the 30S subparticle. When the method of the present invention was carried out without using a reducing agent, spots A, B and C disappeared, but spots B'and C'were found in the vicinity thereof as B and C disappeared. It was
On the other hand, D forms a spot at the same position regardless of the presence or absence of a reducing agent.

Moreover, when the molecular weights of A, B, C, and D were calculated using the two-dimensional mobility, they were 6400, 4900, 8200, and 5900, respectively. The number of copies was measured by ¹⁴ C amino acid labeling, and the values of 0.6, 0.4, 0.3 and 0.1 were obtained, respectively.

EFFECTS OF THE INVENTION Since the method of the present invention is configured as described above, it is possible to accurately and clearly separate and analyze sample components in two-dimensional electrophoresis.

[Brief description of drawings]

1 (a) and 1 (b) are vertical cross-sectional views showing a gel container for zero-dimensional electrophoresis and its use state, and FIGS. 2 (a) and 2 (b) are primary diagrams for carrying out the method of the present invention. FIG. 3 is a perspective view showing the original electrophoresis gel container and the sample gel cover, FIG. 3 is a vertical cross-sectional view showing the one-dimensional electrophoresis gel container and the upper and lower electrode tanks attached thereto, and FIG. 4 is an exploded perspective view thereof. The figure is a perspective view showing various gel container plates for two-dimensional migration separately, and Fig. 6 is a vertical cross section showing a structure in which the gel container plates of Fig. 5 are combined to form a container structure, and the upper and lower electrode chambers are attached to the container structure. FIG. 7, FIG. 7 is an exploded perspective view thereof, FIG. 8 is a graph showing molecular weight-migration distance characteristics obtained by the examples of the present invention, and FIG. 9 is a view showing a separation model of a two-dimensional gel. (1) …… Gel chamber (2) …… Upper (anode) buffer tank (3) …… Lower (cathode) buffer tank (4) …… Running gel A …… Anode B …… Cathode SG …… Sample gel SGC …… Sample gel cover ABV …… Upper (anode) buffer tank CBV …… Lower (cathode) buffer tank LP …… Left container plate MP …… Center container plate RP …… Right container plate D …… Slot between containers G …… Gauze PW …… Flat wedge GS …… Gel spacer H …… Fastener

Claims (1)

[Claims] 1. A one-dimensional gel is prepared in at least one of the rod-shaped gel chambers in a one-dimensional gel producing apparatus in which a plurality of rod-shaped gel chambers each having a rectangular cross section and arranged in parallel are arranged in a two-dimensional electrophoresis method. Then, the sample is electrophoresed in the rod-shaped one-dimensional gel to create a one-dimensional separated gel of the sample having a rectangular cross section, and the one-dimensional separated gel is laid on the upper end of the two-dimensional gel to give a one-dimensional gel. A two-dimensional electrophoresis method characterized in that electrophoresis is performed in a state where the lower surface of the separated gel and the upper end surface of the two-dimensional gel are in close contact with each other without a gap.