JP2005300404A - Electrophoretic gel preparation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrophoretic gel preparation apparatus capable of preventing gel solidification in the middle of preparation in the gel preparation apparatus such as a monomer container. <P>SOLUTION: This electrophoretic gel preparation apparatus is used for preparing polyacrylamide gel for electrophoresis. At least a part thereof is formed from a material having an oxygen transmissivity of 0.5 fm<SP>2</SP>/(s Pa) or more, or preferably from polydimethylsiloxane or a silicone resin. The apparatus can be used suitably as a gel ingredient container, and especially used for preparing gradient gel. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an electrophoresis gel preparation device (hereinafter, also simply referred to as “gel preparation device”), and more particularly to an electrophoresis gel preparation device used when preparing a polyacrylamide gel for electrophoresis.

  As one of high resolution analysis methods for proteins and nucleic acids in the field of life science research, so-called gel electrophoresis is actively used. Examples of the support used in the gel electrophoresis include filter paper, cellulose membrane, agar (agarose), polyacrylamide gel, and the like. Among them, polyacrylamide gel is a synthetic material, and therefore has the advantage that various gels with different properties can be prepared. In particular, it is formed by providing a concentration gradient of polyacrylamide in the gel. Gradient gel has a wide separation range and can be said to have high utility value in the separation of DNA fragments and the like.

  A polyacrylamide gel is produced by mixing an appropriate amount of a crosslinking agent and a polymerization initiator in acrylamide and adding a polymerization accelerator to polymerize the mixture. FIG. 1 shows an apparatus for producing a polyacrylamide gradient gel. In the illustrated apparatus, first, as shown in FIGS. 2 (a) and 2 (b), a substrate 12 having a glass spacer 13 disposed in a gel preparation container 1, a lid 11, a silicone rubber spacer 14, Is installed. In addition, acrylamide monomers having different concentrations are placed in containers 2 and 3, and a crosslinking agent and a polymerization accelerator are introduced immediately before gel preparation. Finally, stirring is performed by the stirrer 4 and the rotor 5 while providing a concentration gradient by opening and closing a valve (not shown) or the like, and each acrylamide is fed from the center of the bottom of the gel preparation container 1 through the gel raw material supply tube 6. By introducing the monomer, a gradient gel having a desired concentration gradient can be produced.

Here, as the monomer containers 2 and 3 for containing the acrylamide monomer, those made of glass, acrylic, polymethyl methacrylate (PMMA) or the like are mainly used from the viewpoint of transparency and high rigidity. Further, for example, Patent Document 1 discloses glass or plastics such as polyethylene, polyacrylate, polyvinyl chloride as the material of the gel support container for electrophoresis.
JP 2002-228632 A ([0008])

  As described above, at the time of preparing the gradient gel, a mixture of acrylamide monomer, a crosslinking agent and a polymerization accelerator having different concentrations is introduced into the monomer containers 2 and 3, respectively, and this is mixed in the gel preparation container 1 at an appropriate ratio. By introducing, a concentration gradient of the gel is formed. However, when a conventional glass or acrylic container is used as the monomer container 2, 3, especially when the preparation time is long due to a large gel capacity, the polyacrylamide on the high concentration side is in the middle of the process. There was a problem that the gel polymerization proceeded and solidified in the monomer container, making it impossible to produce a gradient gel. When the polyacrylamide gel is solidified in the monomer container, the apparatus itself may become unusable. Therefore, it has been desired to solve this problem.

  On the other hand, in order to prevent the gel from solidifying, the entire monomer container has been cooled and the introduction rate of the acrylamide monomer mixture has been increased. However, even if the entire monomer container is cooled, the polymerization rate is only slightly slowed down, and if the amount of acrylamide monomer mixture introduced is increased, a concentration gradient in the gradient gel cannot be created accurately. Neither was an essential solution because problems would arise.

  Accordingly, an object of the present invention is to provide an electrophoresis gel preparation device capable of preventing the gel from solidifying during preparation in a gel preparation device such as a monomer container.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have inhibited the polymerization reaction of acrylamide by applying a high oxygen permeable material as the material of the gel preparation device, and are in the process of preparation in the gel preparation device. It was found that the solidification of the gel can be prevented, and the present invention has been completed.

That is, the electrophoresis gel preparation device of the present invention is an electrophoresis gel preparation device used for preparation of polyacrylamide gel for electrophoresis, and at least a part thereof has an oxygen permeability of 0.5 fm ² / (s. -Pa) It is characterized by being formed of the above material.

  It is known that polymerization of polyacrylamide proceeds by radical reaction. On the other hand, in radical polymerization, generally, when oxygen is present, free radicals are trapped by oxygen and polymerization is inhibited. Therefore, in order to prevent the solidification of polyacrylamide in the gel preparation device, it is considered effective to prevent the polymerization by making oxygen easily enter the gel preparation device.

  Here, the oxygen permeability of the material is determined by the solubility and diffusibility of oxygen. However, since oxygen is nonpolar, the permeability corresponds to the diffusion coefficient, and the influence of the diffusivity becomes large. Therefore, it can be said that a polymer having a weak polarity, no hydrogen bond in the structure, a soft molecular chain, and a low crystallinity is excellent in oxygen permeability.

Examining from such a viewpoint, the oxygen permeability (fm ² / (s · Pa)) of a typical polymer material conventionally used as a material for a gel preparation device such as a monomer container is PMMA: 0.76. × 10 ^-4 , polystyrene (PS, (iso)): 0.019, polycarbonate (PC): 0.011, etc. (see Fumio Ide, “Practical Polymer Material” P220), all of which have low oxygen permeability It is sex. Therefore, in a gel preparation device using these materials, oxygen is difficult to enter inside, and polymerization is not inhibited by oxygen. In the present invention, by using a high oxygen permeable material having an oxygen permeability of 0.5 fm ² / (s · Pa) or more as a material for the gel preparation device, oxygen as a polymerization inhibitor can easily enter the container, This makes it possible to prevent the polyacrylamide gel from solidifying inside the gel preparation device. The gel whose polymerization is inhibited by oxygen is defoamed by making the final container for forming the gel shape from a low oxygen permeable material and stirring the inside of the production apparatus with a stirrer. Thus, the polymerization can be advanced by the polymerization accelerator and finally gelled.

  As the material according to the present invention, polydimethylsiloxane or silicone resin can be suitably used. Moreover, the gel preparation instrument of this invention is suitable as a gel raw material container, for example. Furthermore, the gel preparation device of the present invention can be suitably applied to preparation of a gradient gel.

  According to the gel preparation instrument for electrophoresis of the present invention, solidification of the polyacrylamide gel in the course of preparation in the gel preparation instrument can be prevented without requiring a special operation or a special apparatus. It is possible to prepare a desired gel, for example, a gradient gel having a specific concentration gradient. In the present invention, the device itself is not disabled due to unexpected solidification of the gel.

Hereinafter, embodiments of the present invention will be specifically described.
The present invention is an electrophoresis gel preparation tool used for preparing a polyacrylamide gel for electrophoresis, and is characterized in that at least a part thereof is formed of a predetermined high oxygen permeable material. Thereby, solidification of the polyacrylamide gel in the gel preparation instrument in the middle of preparation can be prevented, and it becomes possible to perform a desired gel preparation process reliably.

  In the present invention, the gel preparation device refers to various components constituting the gel preparation apparatus, and is mixed with a polyacrylamide monomer and a crosslinking agent or a polymerization accelerator and immediately before being introduced into the gel preparation container. The container and the tube corresponding to the supply path of the mixed gel raw material are included. Therefore, since the gel preparation container itself becomes a mold for solidifying the gel, the present invention is not applied. Further, the present invention is not applied to a container that is filled with a monomer, a crosslinking agent, or the like before mixing.

  Specific examples of the gel preparation tool of the present invention include gel raw material containers such as monomer containers 2 and 3 in the gradient gel preparation apparatus shown in FIG. By using the gel preparation device for electrophoresis of the present invention as the monomer container 2 or 3 for containing a mixture of polyacrylamide monomer, crosslinking agent, polymerization accelerator and the like at the time of gradient gel preparation, It is possible to prevent the gel from solidifying before being introduced into the gel preparation container 1. As described above, two types of monomer mixture of low concentration and high concentration are usually used for the preparation of the gradient gel, but the gel preparation device of the present invention is particularly suitable for the high concentration side where polymerization is likely to proceed. It is suitable as a monomer container.

  The gel preparation device of the present invention is also effective when applied to the gel raw material supply tube 6 that connects the gel raw material containers (monomer containers) 2 and 3 and the gel preparation container 1. An effect of inhibiting the progress of the polymerization reaction during the production can be obtained.

The high oxygen-permeable material according to the present invention is a material having an oxygen permeability of 0.5 fm ² / (s · Pa) or more, although it varies depending on the gel concentration used, the amount of polymerization accelerator, and the amount of polymerization initiator. If the oxygen permeability is less than 0.5 fm ² / (s · Pa), the polymerization inhibition effect of polyacrylamide may be insufficient and the preparation of the gel may be hindered. I can't get it. A material having an oxygen permeability of 0.7 to 10 fm ² / (s · Pa) is preferable.

Examples of the high oxygen permeable material that can be used in the present invention include polydimethylsiloxane (PDMS), silicone resin, low density polyethylene, and siloxanyl methacrylate (SiMA) used as a material for oxygen permeable hard contact lenses. ) And methyl methacrylate (MMA) as a main component, cellulose acetate butyrate (CAB), a copolymer mainly composed of SiMA, fluoroalkyl methacrylate (FMA) and methacrylic acid (MMA), siloxanylstyrene and FMA And a copolymer of butyl methacrylate and butyl acrylate, preferably PDMS having an oxygen transmission rate of up to 0.75 fm ² / (s · Pa) (Fumio Ide, “Practical use”). Polymer material " From 220 reference) and oxygen permeability 5.5fm ² / silicone resin (s · Pa).

  Note that the gel preparation device of the present invention is only required to be formed at least partially, preferably entirely using the predetermined high oxygen permeable material. There is no particular limitation.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
(Example)
First, a substrate 22 having a groove (flow path) 23 as shown in FIG. 3 is produced using PDMS (oxygen permeability 0.75 fm ² / (s · Pa)) as a material, and a glass lid 21 is formed on the substrate 22. The device 20 formed by combining these was placed in a gel preparation container 1 made of polystyrene (PS). Next, in order to prepare a 5 to 20% gradient gel in the flow path 23, acrylamide was introduced into the gel preparation container 1 with a concentration gradient using the apparatus shown in FIG. Normally, acrylamide gel solidifies in about 30 minutes, but the gel in the device 20 did not solidify even after 2 hours or more. On the other hand, the gel around the device 20 solidified in about 30 minutes.

  Then, when the gel in the device 20 that did not solidify was placed in a glass container and allowed to stand, it solidified in about 30 minutes.

(Comparative example)
A gradient gel was prepared in the same manner as in Example except that the substrate 22 was formed using a silicone substrate and a resist that did not substantially transmit oxygen. As a result, the gel solidified in about 30 minutes not only around the device 20 but also inside.

  From the results of the above examples and comparative examples, it is possible to inhibit the polymerization of polyacrylamide if the gel preparation device for electrophoresis of the present invention formed of a high oxygen permeable material is used instead of the conventional monomer container. Therefore, it was confirmed that the gel could be reliably produced without solidifying the gel during the production.

  Gel electrophoresis, which is a high resolution analysis method in life science research, is frequently used not only for research but also for testing. Since gels used in this gel electrophoresis method are generally difficult to store for a long period of time, it is necessary to prepare a gel each time for electrophoresis analysis. The preparation of such gels requires specialized knowledge and experience, and has conventionally hindered the widespread use of gel electrophoresis, but the present invention makes it possible to prepare gels easily and reliably. Therefore, it is thought that it can contribute to the spread of further gel electrophoresis.

It is a schematic explanatory drawing which shows the preparation apparatus of a gradient gel. It is a schematic explanatory drawing which shows an example of the device which introduces a gradient gel. It is a schematic explanatory drawing which shows the device which introduce | transduces the gradient gel used in the Example.

Explanation of symbols

1 Gel preparation container 2, 3 Monomer container (gel raw material container)
4 Stirrer 5 Rotor 6 Tube 10, 20 Device 11, 21 Lid 12, 22 Substrate 13 Glass spacer 14 Silicone rubber spacer 23 Groove (flow path)

Claims (4)

An electrophoresis gel preparation device used for preparation of polyacrylamide gel for electrophoresis, wherein at least a part is formed of a material having an oxygen permeability of 0.5 fm ² / (s · Pa) or more. A gel preparation device for electrophoresis.   The gel preparation device for electrophoresis according to claim 1, wherein the material is polydimethylsiloxane or silicone resin.   The gel preparation device for electrophoresis according to claim 1 or 2, which is a gel raw material container. The gel preparation device for electrophoresis according to any one of claims 1 to 3, which is used for preparation of a gradient gel.

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