JPH02309249A - Gel support for two-dimensional electrophoresis device and two-dimensional electrophoresis method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Field of Industrial Application The present invention relates to an electrophoresis apparatus used in fields such as biochemistry and clinical analysis.

B. Prior Art An electrophoresis device is a device that performs analysis and separation by utilizing the phenomenon that when a charged substance in a liquid is placed in an electric field, the charge moves toward an electrode with a sign opposite to that of the charged substance. The speed of movement is affected by the strength of the electric field, the pH of the medium, ionic strength, dielectric constant, temperature, etc., but if these conditions are constant, it will depend on the specific values of the sample material, such as the amount of charge, size, shape, and viscosity of the solution. Since it is determined by Especially in bio-related technology, starting from small ions, biopolymers such as proteins and nucleic acids,
Since it handles various charged particles, including organelles and cells, it is highly effective and is used for the separation and analysis of various substances, such as DNA base sequencing and analysis of trace proteins.

Gel electrophoresis is currently the mainstream method, as it has a superior gel molecular sieving effect and can provide high resolution compared to other electrophoresis methods. There are two types of gel electrophoresis devices: tube gel electrophoresis devices, which are made between two parallel glass or plastic plates, and slab gel electrophoresis devices, which are made between two parallel glass or plastic plates.

Secondary dead-eye phoresis using the above-mentioned slab gel electrophoresis for first-dimensional phoresis has conventionally been carried out as follows.

■ Create a slab gel by injecting the gel solution using two glass plates and a spacer. ■ After the first-dimensional electrophoresis is complete, remove the first-dimensional gel from the tube or cut only the loons from the slab and take it out. Take it out, and (1) place it at a predetermined position on a secondary dead gel to perform secondary dead gel migration.

Problems to be Solved by the C0 Invention The above-mentioned conventional techniques require considerable skill in handling and preparing gels, and are difficult to operate easily. As a result, there was a problem that the reproducibility and separation ability of electrophoretic separation were affected, and stable operation could not be performed.

In order to solve the problems of the prior art described above, the present invention allows a first-dimensional gel to migrate on a bendable base material, then bends the base material, and joins the first-dimensional gel to a second dead-size gel. By performing electrophoresis while fixing and holding the first-dimensional gel and the second dead gel, it is possible to prepare a ready-made gel for two-dimensional electrophoresis, thereby expanding the scope of the research field of the two-dimensional electrophoresis method itself. The aim is to eliminate the complicated work involved in creating the system. A second object of the present invention is to easily and reproducibly bond a first-dimensional gel and a second-dimensional dead gel. A third object of the present invention is to improve the electrophoretic separation ability by controlling the temperature during electrophoresis.

00Means to solve problems In order to achieve the above object, the present invention has the following configuration.

That is, the present invention provides (1) a gel support for a two-dimensional electrophoresis device, in which a first-dimensional gel and a second dead gel are placed separately at positions sandwiching a bending point of a bendable support; After performing electrophoresis with the first-dimensional gel of the support in item (1) above, the first-dimensional gel migration part is bent and joined to the second dead-mesh gel migration part, and the first-dimensional gel is It is characterized in that the secondary dead gel is fixed and supported while the secondary dead gel is fixed and supported, and the secondary dead gel is fixed and supported. Further, the bent portion may be a hinge mechanism or the like, or in the case of a film or sheet, it may be bent depending on its own characteristics.

The temperature control mechanism of the present invention may be of any known type. Examples include circulation of cooling water and temperature control using a Peltier device.

The characteristic requirements of the present invention are listed below.

(1) It has a means for fixing and holding the electrophoresis gel on the base material.

■ The structure is such that the base material is bent between the first-dimensional gel and the second-dimensional dead gel.

(3) The base material has a structure that allows temperature control.

Since the E1 action gel is fixed and held on the base material, it is easy to handle. Furthermore, the joining of the first-dimensional gel and the second-dimensional dead gel can be easily and reproducibly performed. In addition, since the temperature of the base material can be controlled, it is possible to control the cooling temperature during migration.

F. Example The present invention will be explained in more detail below using Examples.

Example 1 1 to 5 show one embodiment of the present invention.

Number 1 in FIG. 1 is a diagram of the entire electrophoresis apparatus.

Ready-made gels for two-dimensional electrophoresis are supplied in vacuum-packed form. This device has a structure in which base materials 2 and 3 are separated by a fulcrum 4 and can be bent freely. Base material 2
, 3 are each structured to form one circulation path, and by connecting piping from the outside, a circulation flow path for temperature adjustment is formed. A first-dimensional gel is fixed and held on the base material 2, and a two-dimensional mouth gel is fixed and held on the base material 3 so as to be parallel to each other.

Number 7 is a flow port for circulating fluid for temperature adjustment.

FIG. 2 shows a schematic diagram of first-dimensional migration. Install + and - electrode tanks and fill them with electrode solution. Cross-link with the first-dimensional gel using filter paper, sponge, etc., and bring the gel into contact with the electrode solution. Then, a voltage is applied between the electrodes to perform migration. At this time, a circulating fluid is caused to flow through the substrate 2, and the cooling temperature during migration is controlled by an external temperature control device (not shown).

The sample can be added directly onto the first-dimensional gel.

Next, as shown in FIG. 4 and FIG. Do it by folding it. The bonded state may be either shown in FIG. 4 or FIG. 5.

FIG. 3 shows a schematic diagram of secondary dead eye migration. As in the case of the first dimension, set up + and - electrode tanks, fill each with electrode solution,
Electrophoresis is performed by cross-linking the electrode solution and gel with filter paper, etc., and applying a voltage between the electrodes. At this time, circulating fluid is flowed into the base material 3,
Cooling temperature is controlled during migration using an external temperature control device.

As described above, according to the present invention, it was confirmed that a gel for two-dimensional electrophoresis can be made ready-made, the operation is simple, and the joining of the first-dimensional gel and the second dead-eye gel can be performed easily and with good reproducibility. .

Furthermore, it was confirmed that the electrophoretic separation ability could be improved by adjusting the temperature during electrophoresis.

Embodiment 2 Another embodiment of the present invention will be described below with reference to FIGS. 6 to 10.

As the base material, a film base material 8 made of polyester or the like is used, and the surface of the film is treated to form a covalent bond with acrylamide (eg, silane coupling treatment). The back surface of the film base material 8 is treated with an adhesive material. The first-dimensional gel 5 and the second-dimensional gel 6 are fixed and held on the film base material 8 facing each other with a cut (slit) 9 as a boundary, as shown in FIG.

Further, the film base material 8 has a structure in which it can be bent at cuts (slits) 9.

FIGS. 8 and 9 schematically show the first-dimensional migration.

10 is a temperature control plate made of a highly thermally conductive material treated with an insulating film. Examples of highly thermally conductive materials include metal materials such as aluminum.

Examples of insulating film treatments include pasting a film or coating.

The lid 11 has a structure including electrode tanks 12 and 13, and a filter 14 (made of porous resin or ceramic, for example) for bringing the electrode solution and gel into contact with each other. Temperature adjustment is performed using a temperature control element such as a Peltier element 15. 16 is a sample port.

FIG. 10 shows an outline of secondary dead eye migration. The lid 21 also has a structure including electrode vessels 22, 23 and a filter 24 as in FIG.

Furthermore, although not shown, temperature control, voltage, current,
Monitoring and controlling the time etc. is done by a computer (CPU).
It may also be done by

The process of the embodiment shown in FIGS. 6 to 10 will be explained below.

(1) Peel off the film on the back of the substrate of the electrophoresis gel and paste it on the temperature control plate at a predetermined position with the adhesive side covered. (FIG. 7(a)).

■ Set parameters such as voltage, current, time, etc.
The first-dimensional lid 11 is folded down, the lid is placed over the gel, and the electrode solution is filled to start the first-dimensional electrophoresis. The sample is placed directly onto the gel from the sample boat 16 using a microsyringe. During electrophoresis, the cooling temperature of the plate 10 is controlled by the Peltier device 15 or the like.

(3) After the first-dimensional electrophoresis is completed, as shown in Figure 7(b), fold the first-dimensional gel part and join it on top of the second dead-mesh gel, overlap it, fix the hole part with a pin, etc., and join it. Create a state.

(4) The lid 21 for secondary dead eye is brought down, a lid is placed on the gel, and the electrode solution is filled to perform secondary dead eye migration.

(5) After the electrophoresis is completed, the gel is peeled off from the temperature control plate along with the base material, and fixed, stained, and processed.

[Modification] As an application example of the present invention, a fully automated two-dimensional electrophoresis apparatus can be created by automating the feeding of the electrode solution (Buffer), the operation and sampling of the lid, and the like.

G0 Effects of the Invention The present invention allows a first-dimensional gel to migrate on a bendable base material, then bends the base material to bond the first-dimensional gel to a second dead-eye gel, and then separates the first-dimensional gel from the first-dimensional gel. Since electrophoresis is performed while the secondary dead gel is fixed and held, the gel for two-dimensional electrophoresis can be made ready-made, and the operation is easy. This could be done easily and with good reproducibility. Furthermore, by controlling the temperature during electrophoresis, it was possible to improve the electrophoretic separation ability.

More specifically, it is as follows.

(b) By making the gel for the first dimension and the second dead eye gel fixed and held on the base material as a ready-made product, the cumbersome work at the time of production is eliminated. ■Gel becomes easier to handle.

(b) The bending of the base material facilitates the bonding of the first-dimensional gel and the second-dimensional dead gel, and also improves the reproducibility of the bonding position.

(c) Since the base material has a structure that allows temperature control, it is possible to prevent a decrease in separation ability due to distortion of the zone due to thermal convection during migration.

[Brief explanation of the drawing]

1 to 5 show one embodiment of the present invention. 6-10 show another embodiment of the invention. 1: Electrophoresis device 2.3: Base material 4: Fulcrum 5. Gel for first dimension 6: Gel for second dimension 8: Film base material 10: Temperature control plate
11: Lid 12. 13: Electrode tank 14: Filter 16: Sample port 1: Electrophoresis device fi1 Figure 2511 3rd
Figure 4 Figure 5 Figure 6

Claims (2)

[Claims] (1) A gel support for a two-dimensional electrophoresis device, in which a first-dimensional gel and a second-dimensional gel are placed separately at positions sandwiching a bending point of a bendable support. (2) After performing electrophoresis using the first-dimensional gel of the support of claim 1, the first-dimensional gel migration part is bent and joined to the second-dimensional gel migration part, and the first-dimensional gel and second-dimensional gel migration part are joined together. A two-dimensional electrophoresis method characterized by performing second-dimensional electrophoresis with a gel fixed and supported.