JP5287568B2 - Formation method of gel separation medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for forming a gel separation medium having the process for filling a container for forming a gel with two gel precursor solutions different in composition, making the interface between the two gels horizontal without disturbing the same and capable of simply forming the gels with good reproducibility. <P>SOLUTION: In the method for forming the gel separation medium configured to fill the container 3 for forming the gel with the two gel precursor solutions different in composition while forming the interface between two gels horizontally, the container 3 is filled with the first gel precursor solution and a solvent having a boiling point lower than that of the solvent constituting the gel precursor solutions is subsequently superposed before filling the container with the two gel precursor solutions. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a method for forming a gel separation medium, for example, a method for forming a gel separation medium for electrophoresis used in two-dimensional electrophoresis for protein separation.

  At the end of the Human Genome Project, protein profiling research is actively underway. Further, not only in the field of such research but also in the field of medical care, there is a demand for more appropriate diagnostic techniques by obtaining knowledge from analysis of patient proteins.

  The most promising method for separating proteins is separation by two-dimensional electrophoresis. This is a combination of separation methods based on the two properties of charge and molecular weight of individual proteins.

  The most widely used method for separating proteins by molecular weight is called SDS-PAGE (Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis), and SDS (dodecyl sulfate) is used in an aqueous gel of acrylamide polymer with a cross-linked structure. Sodium), a charged protein is introduced by treatment and electrophoresed. At this time, since polyacrylamide has a crosslinked structure, it has an effect as a molecular sieve, and proteins are separated according to molecular weight.

  Separation media in SDS-PAGE are generally gels composed of polyacrylamide aqueous gels with hardness and molecular sieving effects suitable for protein electrophoresis (hereinafter referred to as “separation gels”) and lower than separation gels. The gel is composed of a polyacrylamide aqueous gel having a concentration (hereinafter referred to as “concentrated gel”). The two gels are in contact and the protein is first introduced into the concentrated gel and then through the concentrated gel and then into the separation gel. At this time, in order to perform accurate separation, it is important that the interface between the two gels is formed horizontally.

  The polyacrylamide aqueous gel used at this time is composed of an acrylamide monomer and a crosslinking agent such as N, N′-methylenebisacrylamide (BIS), a buffer solution such as an aqueous trishydroxymethylaminoethane-HCl solution, ammonium persulfate in a mixture of water, A solution obtained by introducing a radical polymerization initiator such as N, N, N ′, N′-tetramethylethylenediamine (referred to as a gel precursor solution) is filled in a gel-forming container and gelled as the polymerization proceeds. It is produced by this.

  In order for such protein separation methods using polyacrylamide aqueous gels to become widespread in clinical settings, cassette-like products that are easy for analysts to use from the viewpoint of reproducibility and complexity of work. In addition, it is desired that a precast gel cassette or the like in which a gel is previously formed is produced with good reproducibility and at low cost.

  Usually, the separation medium is formed in SDS-PAGE by filling the container for gel formation with the separation gel precursor and then overlaying it with, for example, water as a layered solvent, and forming the gel in the presence of the layered water. Then, the layered solvent is removed and the concentrated gel precursor solution is filled (Non-Patent Document 1). The role of water in this case is to form the gel interface horizontally and prevent contact with oxygen that inhibits radical polymerization.

  However, although the gel prepared by the above method is formed with a favorable interface between the separation gel and the concentrated gel, it is necessary to remove the layered water by tilting the cassette or sucking out with a dropper before filling the concentrated gel precursor solution. Therefore, the process is complicated, and careful operation is required so as not to disturb the interface. Also, until the polymerization has progressed and gelled sufficiently, the superposed water cannot be removed and the process cannot proceed to the next step. These problems are difficult to automate and are disadvantageous when considering industrial mass production.

  A method of filling the concentrated gel precursor solution without waiting for gel formation after filling the separated gel precursor solution is also conceivable. In that case, although the time required for gel preparation can be shortened, it is expected that the interface between the separation gel and the concentrated gel is disturbed and mixed. These are concerns as problems of productivity and reproducibility in mass production.

Protein Experiment Handbook, Yodosha, August 15, 2003, p58-59

  Therefore, the problem in the present invention is that the interface between the two types of gels is not disturbed in the method of forming a gel separation medium having a step of filling two types of gel precursor solutions having different compositions in a container for forming a gel. It is an object of the present invention to provide a method for forming a gel separation medium that is horizontal and can be easily and reproducibly formed.

The invention according to claim 1 is a method for forming a gel separation medium, in which two types of gel precursor solutions having different compositions are filled in a container for forming a gel, and an interface between the two gels is horizontally formed. There,
After the first kind of gel precursor solution is filled in the container, a solvent having a boiling point lower by 30 ° C. or more than the solvent constituting the gel precursor solution is overlaid, and then the second kind of gel precursor solution is filled. This is a method for forming a gel separation medium.
A second aspect of the present invention is the formation of the gel separation medium according to the first aspect, wherein after the first type gel precursor solution is filled, the boiling point of the solvent to be layered is 70 ° C. or less. Is the law.
The invention according to claim 3 is the method for forming a gel separation medium according to claim 1 or 2 , characterized in that, after the first kind of gel precursor solution is filled, the layered solvent is hydrophobic. It is.
The invention according to claim 4, before the filling of the two eyes of the gel precursor solution, the formation of gel separation medium according to claim 1, wherein the solvent is characterized in that it is vaporized Is the law.
In the invention according to claim 5 , the solvent layered after filling the first type gel precursor solution is diethyl ether, ethyl propyl ether, dipropyl ether, n-pentane, isopentane, n-hexane, 2- It is at least 1 type selected from the group which consists of methylpentane, 3-methylpentane, and 2, 2- dimethylbutane, It is the formation method of the gel separation medium in any one of Claims 1-4 characterized by the above-mentioned. .

  In the method for forming a gel separation medium of the present invention, after the first kind of gel precursor solution is filled in the container, a solvent having a boiling point lower than that of the solvent constituting the gel precursor solution is overlaid, and then the second kind of gel Since it is characterized by filling the precursor solution, the layered solvent is removed from the container for gel formation by vaporizing, and unlike conventional water layering, it is easy without removing the special layering solvent. A simple gel can be made. Therefore, industrial mass production can be facilitated. Furthermore, in this method, since it is not necessary to apply a special force to the gel interface, the gel interface is not damaged. Further, in this method, the time until the multi-layer solvent is completely vaporized can be controlled by the amount of the multi-layer solvent, the boiling point, or the heating of the container. For this reason, it is possible to set so that the multi-layer solvent exists in accordance with the time until the polymerization proceeds and the gel is formed.

(A) is a figure of the structural member of the cassette used for description of embodiment of this invention, (B) is a figure of the cassette used for description of embodiment of this invention.

  Examples of the container for forming a gel that can be used in the present invention include the cassette shown in FIG. FIG. 1A is a diagram of the components of the cassette used to describe the embodiment of the present invention, and FIG. 1B is a diagram of the cassette used to describe the embodiment of the present invention. The cassette constituting member shown in FIG. 1 (A) has a gel precursor solution filling port 5 as shown in FIG. 1 (B) by joining the cassette constituting portion 1 having a recess and the cassette constituting portion 2 by an appropriate means. A gel-forming container 3 in which is formed is obtained. The buffer contact port 4 is used after being sealed with a sealing material. This sealing material may be a film having a hot melt property or a cap made of a resin capable of sealing the opening.

  The material of the cassette component 1 and the cassette component 2 may be any material that does not have a problem in gel formation and does not have a problem in electrophoresis, and is preferably an acrylic resin such as polymethyl methacrylate resin. It may be made of resin or glass.

Hereinafter, the filling method will be described. Embodiment 1 below is a method of filling a cassette with the first type of gel precursor solution, then overlaying the solvent, removing the layered solvent by volatilization, and then filling the second type of gel precursor solution. is there.
Here, when the present invention is used for electrophoresis, for example, SDS-PAGE, the first kind of gel precursor solution is a separated gel precursor solution, and the second kind of gel precursor solution is a concentrated gel precursor solution. After gelation, the separated gel precursor solution and the concentrated gel precursor solution become a separated gel and a concentrated gel, respectively.
In addition, the description regarding the boiling point of the liquid in this invention shall be in 1 atmosphere.

Embodiment 1
After the separation gel is filled in the cassette shown in FIG. 1 (B) from the gel precursor solution filling port 5, a solvent having a boiling point of 30 ° C. or more lower than that of the solvent constituting the first kind of gel precursor solution is overlaid. In SDS-PAGE, the solvent constituting the first kind of gel precursor solution (separated gel precursor solution) and the second kind of gel precursor solution (concentrated gel precursor solution) is water, and the boiling point at 1 atm is 100 ° C. is there. In this case, the boiling point of the multilayer solvent is preferably 70 ° C. or lower. Specifically, at least one selected from the group consisting of diethyl ether, ethylpropyl ether, dipropyl ether, n-pentane, isopentane, n-hexane, 2-methylpentane, 3-methylpentane, and 2,2-dimethylbutane. Overlay the solvent. You may mix and use a multilayer solvent.
At this time, as in the case where conventional water is used as the multilayer solvent, a horizontal interface is formed at the contact portion between the first type gel precursor solution and the multilayer solvent. By using a hydrophobic solvent as a multilayer solvent in the present invention, for example, in SDS-PAGE, mixing with the separation gel precursor solution is prevented, and a good interface state can be maintained.

  Then, after the first type gel precursor solution is sufficiently gelled, the layered solvent is vaporized and removed from the cassette. In this case, the solvent may be vaporized by spontaneously vaporizing the solvent, heating the cassette, or creating a reduced pressure environment. If the boiling point of the layered solvent is lower by 30 ° C. or more than the boiling point of the solvent constituting the gel precursor solution, it is preferable because the layered solvent can be removed by heating or reduced pressure without adversely affecting the gel preparation.

  After removing the multilayer solvent, the second gel precursor solution is filled. Compared with the conventional method using water as the overlay solvent, this method fills the second gel precursor solution without removing the overlay solvent or tilting the cassette and discarding the solvent. It becomes possible to do.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not restrict | limited to the following example.

  A separated gel precursor solution having the composition shown in Table 1 below was filled from the gel precursor solution filling port 5 into a cassette having a structure similar to that of the gel forming container 3 having a gel forming region thickness of 1 mm and a capacity of 5 ml. Thereafter, 0.4 ml of diethyl ether was layered from the gel precursor solution filling port 5. The boiling point of diethyl ether is about 35 ° C.

Table 1
Acrylamide aqueous solution (solid content concentration 30 w / v%): 1.334 ml
(The solid content of the acrylamide aqueous solution is an aqueous solution of a mixture in which the weight ratio of acrylamide to BIS is 29.2: 0.8.)
1.5 M Tris-HCl aqueous solution (pH 8.8): 1.000 ml
Ammonium persulfate aqueous solution (solid concentration: 10 w / v%): 0.040 ml
Tetramethylethylenediamine: 0.004ml
Distilled water: 1.622 ml

  At this time, diethyl ether was clearly separated from the separated gel precursor solution, and the interface between the separated gel precursor solution and diethyl ether was formed horizontally. Diethyl ether can no longer be seen in about 5 minutes at room temperature, and it has been confirmed that the separated gel precursor solution has a hardness that does not cause a problem with filling of the concentrated gel precursor solution over the course of that time. .

  As described above, according to the present invention, in the method of forming a gel separation medium having a step of filling two types of gel precursor solutions having different compositions in a container for forming a gel, The interface between the gels is horizontal without being disturbed, and the gel can be easily and reproducibly formed. Therefore, if the method of this invention is utilized, it will be useful, for example for the research for protein separation, or preparation of the precast gel used for a clinical use.

DESCRIPTION OF SYMBOLS 1 ... Cassette structure part 2 ... Cassette structure part 3 ... Gel formation container 4 ... Buffer contact port 5 ... Gel precursor solution filling port

Claims (5)

A method for forming a gel separation medium in which a container for forming a gel is filled with two kinds of gel precursor solutions having different compositions, and an interface between the two gels is horizontally formed,
After the first kind of gel precursor solution is filled in the container, a solvent having a boiling point lower by 30 ° C. or more than the solvent constituting the gel precursor solution is overlaid, and then the second kind of gel precursor solution is filled. A method for forming a gel separation medium.   2. The method for forming a gel separation medium according to claim 1, wherein after the first type of gel precursor solution is filled, the boiling point of the solvent to be layered is 70 ° C. or less. The method for forming a gel separation medium according to claim 1 or 2 , wherein after the first type gel precursor solution is filled, the solvent to be layered is hydrophobic. The method for forming a gel separation medium according to any one of claims 1 to 3 , wherein the solvent is vaporized before filling the second kind of gel precursor solution. Solvents layered after filling the first kind of gel precursor solution are diethyl ether, ethylpropyl ether, dipropyl ether, n-pentane, isopentane, n-hexane, 2-methylpentane, 3-methylpentane and 2 The method for forming a gel separation medium according to claim 1 , wherein the gel separation medium is at least one selected from the group consisting of 1,2-dimethylbutane.

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