JP2005241477A - Gel for electrophoresis and its utilization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide gel for electrophoresis capable of recovering a sample such as protein highly effectively capable of easy production, and to provide its utilization. <P>SOLUTION: The gel 10 for electrophoresis used for extracting and recovering the sample from the gel separated by the electrophoresis, wherein the sample inputting part 11 for inputting the sample and the sample recovering part 12 for recovering the separated sample are formed. By the gel 10 in which the thickness D2 of the gel at the sample recovering part 12 is made thinner than the thickness D1 of the gel at the sample inputting part 11, the sample such as the protein, can be recovered with a high yield. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a gel for electrophoresis and use thereof, and more particularly to a gel for electrophoresis suitable for analysis and recovery of biological samples such as proteins and peptides and use thereof.

  Conventionally, gel electrophoresis is commonly used in the field of molecular biology. Gel electrophoresis is an analytical means for separating biomolecules by a kind of molecular sieving effect. That is, when electrophoresis is performed using a gel having an appropriate gel concentration, the migration speed varies depending on the size of the molecule, so that excellent separation can be obtained. For this reason, for example, it is commonly used for purity analysis of samples for analysis and purification of mixtures of proteins and other biopolymers.

  Among the gel electrophoresis methods, acrylamide gel electrophoresis, which is a kind of zone electrophoresis using polyacrylamide gel as a support (carrier), is often used. Briefly describing the acrylamide gel electrophoresis method, a gel is prepared by adding a crosslinking agent N, N'-methylenebisacrylamide to acrylamide and polymerizing the gel. These gels have been devised to have a flat plate shape, a thin cylindrical shape, etc., but a flat plate shape is often used. Hereinafter, the flat acrylamide gel electrophoresis method will be described in detail as an example.

  Two glass substrates are arranged at a predetermined interval, and acrylamide injected between these two glass substrates is polymerized to produce an electrophoresis gel. At the upper end of the gel is a recess for injecting a sample to be analyzed, and this recess is generally called a well. The shape and size of the well are determined by the shape and size of a “mold” called a comb used when producing the gel. When the comb is set before gelation by polymerization of acrylamide and the comb is pulled out after gelation, a well is formed in the pulled portion.

  While the gel is sandwiched between the glass substrates, the end (upper end) of the gel forming the well and the other end (lower end) of the gel are immersed and brought into contact with the buffer solution. Since the sample to be analyzed is a solution, the sample solution can be submerged in the well by making the specific gravity of the sample solution larger than the specific gravity of the buffer in which the gel is immersed in advance. After adding the sample in this manner, a voltage is applied to both ends of the gel via the buffer, thereby introducing the protein in the sample solution into the gel.

  In the case of acrylamide gel electrophoresis, the acrylamide concentration and the buffer pH are adjusted so that the protein sample is concentrated in the gel portion close to the well. The gel portion thus adjusted is referred to as a concentrated gel (stacking gel). After the protein sample injected into the well is concentrated in this part, it is led to a part called a separation gel and separated by the difference in migration speed depending on the molecular size. Finally, the proteins separated in the gel by electrophoresis are visualized by staining or the like.

  The general structure of the gel used in such acrylamide gel electrophoresis is as follows: the thickness of the acrylamide gel formed between two glass substrates: 1 to 2 millimeters, inserted into an acrylamide solution to form a well Comb thickness: 1 to 2 millimeters. The general well size is 2 to 5 millimeters in width (perpendicular to the migration direction) and 3 to 15 millimeters in depth (parallel to the migration direction). The injection amount of the sample solution to be analyzed is a very small amount of several microliters to several tens of microliters.

Furthermore, various gel gels for electrophoresis have been developed by improving the general gel structure described above. For example, Patent Document 1 discloses a technique using a flat electrophoresis gel composed of a concentrated gel having a thickness of 50 μm to 350 μm and a separated gel having a thickness of 50 μm or less in order to increase the separation efficiency of the sample. Yes. Patent Document 2 discloses the use of an electrophoretic separation gel having two or more closely-adhered electrophoretic slab gel layers in order to perform staining and separation / purification based on the same electrophoretic pattern. Has been. Patent Document 3 discloses an electrophoresis gel that uses a wedge-shaped comb in order to facilitate sample injection.
JP 2001-133438 A (publication date: May 18, 2001) Japanese Patent Laid-Open No. 1-96550 (Publication date: April 14, 1989) Patent Publication No. 2620504 (Registration Date: March 11, 1997, Corresponding Publication: Japanese Patent Laid-Open No. 6-229983 (Publication Date: August 19, 1994))

  By the way, in recent years, not only proteins are separated simply by gel electrophoresis, but one step further, the proteins separated by gel electrophoresis can be recovered from the gel, or peptides that are protein hydrolysates can be obtained. Recovering from gels is becoming increasingly popular. For example, while maintaining the protein separated by electrophoresis in a gel, a gel piece containing the protein can be cut out with a knife or the like, and the protein can be recovered from the gel by a technique such as electrical or diffusion. In addition, a gel piece holding a protein can be immersed in a proteolytic enzyme solution, the protein is cut into peptide fragments in the gel, and then the peptide generated in the gel can be recovered from the gel by diffusion or the like.

  However, the configuration of the conventional gel for electrophoresis has a problem that the recovery efficiency is low when recovering proteins separated by electrophoresis. This is because when protein or the like is recovered from the gel, the protein or the like moves from the inside of the gel to the outside of the gel, but it is considered that the recovery efficiency and the recovery speed decrease as the moving distance increases.

  When recovering protein or the like from the gel, it is sufficient to reduce the volume of the gel in which the protein is embedded, and this can be realized by thinning the gel. However, in a general gel, if the gel is thinned, the volume of the sample that can be loaded is reduced accordingly. For this reason, it becomes difficult to put a sufficient amount of protein into a gel for a dilute protein solution, which makes analysis impossible.

  In addition, Patent Documents 1 to 3 disclose a technique for increasing the separation efficiency, a technique for making the same electrophoresis pattern in the staining gel and the separation gel, and a technique for facilitating sample injection, respectively. However, none of these prior art documents disclose or suggest the problem of low efficiency when recovering a protein from a gel. Moreover, in the technique of patent document 3, a general purpose gel preparation apparatus cannot be used, and it is very disadvantageous.

  As described above, until now, little has been considered about attempts and problems to efficiently recover proteins from gels. Therefore, the present inventors independently found this problem and tried to solve it. That is, there has been a strong demand for the development of an electrophoresis gel capable of recovering biological samples such as proteins and peptides from the electrophoresis gel with high efficiency and its use.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide an electrophoresis gel that can be easily produced and can efficiently collect a sample such as a protein, and use thereof. Is to provide.

  As a result of intensive studies in view of the above problems, the present inventors have used an electrophoresis gel in which the gel thickness of the portion into which the sample is introduced is increased and the gel thickness of the portion where the separated protein is to be recovered is reduced. When the protein was recovered after electrophoresis, it was found that the recovery efficiency was remarkably improved as compared with the conventional general gel for electrophoresis, and the present invention was completed.

  That is, the electrophoresis gel according to the present invention is an electrophoresis gel used for extracting and recovering a sample separated by electrophoresis from the gel, and separated from a sample loading portion for loading the sample. A sample recovery part for recovering the sample, and the thickness of the gel in the sample recovery part is thinner than the thickness of the gel in the sample input part.

  In the electrophoresis gel, it is preferable that the electrophoresis gel includes a concentrated gel and a separation gel, and the sample recovery unit is formed at least in part of the separation gel.

  In the electrophoresis gel, the thickness of the sample recovery part is preferably 0.1 mm or more and 0.75 mm or less.

  Further, in the electrophoresis gel, the electrophoresis gel is sandwiched and held between two substrates, and the substrate and the sample recovery are caused by the difference in thickness between the sample loading part and the sample recovery part. It is preferable that a space filling means is provided in a gap generated between the two portions.

  In the method for producing an electrophoresis gel according to the present invention, the space filling means is characterized in that a flat thin layer or two or more thin flat layers are laminated.

  Moreover, it is preferable that the space filling means and the sample recovery unit are fixed. Here, the fixation means that the gel for electrophoresis is removed from the substrate (for example, a glass substrate) and is fixed to the extent that it can be handled as a unit when performing a staining process or a gel cutting process (sample recovery process). It should be.

  The sample is preferably a protein, peptide, DNA, or RNA.

  Further, in the method for producing an electrophoresis gel, the method for producing an electrophoresis gel described above, wherein at least two substrates are arranged so as to face each other at a predetermined interval, A space filling means is provided in the gap provided between the two substrates so as to be in contact with one of the substrates and not to be in contact with the other substrate. By injecting the gel composition, the sample collection part is formed in a gap between the substrate not in contact with the space filling means and the space filling means.

  The space filling means is preferably a flat plate-like thin layer or a laminate of two or more flat plate-like thin layers.

  Moreover, the gel for electrophoresis manufactured by the manufacturing method of the said gel for electrophoresis may be contained in this invention.

  The electrophoresis gel manufacturing kit according to the present invention is a manufacturing kit for manufacturing the electrophoresis gel, and is characterized by comprising at least space filling means.

Moreover, in the said manufacturing kit, it is preferable to have at least 1 among the members of the following (a)-(c).
(A) At least two substrates (b) Gap means for arranging two substrates to face each other with a predetermined gap (c) Gel material The electrophoretic device according to the present invention is It is characterized by having an electrophoresis gel as a support.

  In addition, the sample recovery method according to the present invention is characterized in that electrophoresis is performed using any one of the above-described electrophoresis gels, the sample is separated, and then the sample is extracted and recovered from the sample recovery unit.

  As described above, according to the gel for electrophoresis according to the present invention, the volume of the gel in the sample recovery unit is reduced. For this reason, when a sample (for example, protein) separated as a result of electrophoresis is extracted and recovered by the sample recovery unit, the sample moving distance when moving the sample from the gel to the outside of the gel is reduced. can do. Therefore, the sample can be recovered with high yield from the gel piece containing the sample after electrophoresis. Furthermore, since it can collect | recover efficiently, there exists an effect that a sample can be collect | recovered with high purity.

  The present invention provides an electrophoresis gel capable of efficiently recovering a sample separated by electrophoresis from the gel, a method of using the gel, and the like. Therefore, the use of the electrophoresis gel according to the present invention is very useful for analysis of biological samples such as peptides and proteins. In the following description, first, an electrophoresis gel according to the present invention (sometimes simply referred to as “gel” for convenience of description) will be described, followed by a method for manufacturing an electrophoresis gel, a manufacturing kit, an electrophoresis device, The sample collection method will be described in order.

[1] Electrophoresis gel The electrophoresis gel according to the present invention includes a sample input portion for inputting a sample and a sample recovery portion for recovering the separated sample. The thickness of the gel in the portion is formed to be thinner than the thickness of the gel in the sample loading portion. That is, in the electrophoresis gel according to the present invention, compared to a region (for example, a portion where a well is formed) into which a sample (for example, protein or the like) is injected (injected), a region for collecting a separated sample (for example, The separation gel is formed so that the thickness of at least a part thereof is reduced. Then, the sample separated by the thinly formed gel portion is cut out together with the gel using the band as an index, and used to extract and collect the sample from the sample-containing gel piece.

  A specific configuration of the electrophoresis gel according to the present embodiment will be described below with reference to FIG. FIG. 1A is a diagram schematically showing a planar configuration of the electrophoresis gel according to the present embodiment, and FIG. 1B is a diagram illustrating the electrophoresis gel of FIG. It is a figure which shows typically the structure of the cross section at the time of cut | disconnecting, FIG.1 (c) typically shows the other structure of the cross section when the gel for electrophoresis of (a) is cut | disconnected by the L1-L1 line | wire. FIG.

  As shown in FIG. 1A, the electrophoresis gel 10 according to the present embodiment includes a sample input unit 11 and a sample recovery unit 12. A well 18 for injecting a sample for each lane is formed in the sample insertion portion 11. In this embodiment, three wells are formed.

  The sample loading part 11 is an area provided for loading or injecting a sample into the electrophoresis gel. For this reason, the specific configuration such as the material, concentration, size, and shape is not particularly limited. For example, there is a region where a well used in a conventionally known electrophoresis gel is formed. preferable. The specific structure such as the number and size of the wells is naturally not limited, but may be a well formed using a comb used in a commercially available gel preparation kit for electrophoresis. Further, for example, when a commercially available electrophoresis gel preparation kit is used, the thickness of the sample insertion portion is 1 mm to 2 mm as shown in the examples described later.

  The sample collection unit 12 is an area for collecting a sample separated by electrophoresis based on the size of molecules. For this reason, as with the sample loading unit 11, the specific configuration such as the material, concentration, size, shape, position, etc. is not particularly limited. For example, at least a part of a conventionally known separation gel It can be used as a sample recovery unit. The position at which the sample collection unit 12 is provided can be appropriately set according to the position at which the sample is collected. For example, if you want to collect a sample with a relatively low molecular weight, you can place it near the lower end of the electrophoresis gel. If you want to collect a sample with a relatively high molecular weight, run the sample from the center of the gel to the vicinity of the upper end. It is also possible to provide a collection unit.

  Further, as shown in FIG. 1B, the gel thickness D2 of the sample recovery unit 12 is formed thinner than the gel thickness D1 of the sample input unit 11. In FIG. 1 (b), the sample collection unit 12 is cut at a substantially right angle and is formed so as to be thinned rapidly. For example, as shown in FIG. There may be. Moreover, although not illustrated, the shape which becomes thin so that an arc may be gently drawn may be sufficient.

  In addition, “the gel thickness D2 in the sample recovery unit 12 is formed thinner than the gel thickness D1 in the sample input unit 11” means that the gel thickness D2 of the sample recovery unit 12 is less than the sample input unit 11. As long as it is thinner than the thickness D1 of 11 gels, it can be set as appropriate as to how thin it should be. For example, in the present embodiment, the gel thickness D1 of the sample loading unit 11 is 1 mm, but the thickness of the gel D2 of the sample recovery unit 12 is preferably 0.1 mm to 0.75 mm. As shown in the examples described later, it may be technically difficult to produce a gel thinner than 0.1 mm, and if it is too thin, the operability when collecting a sample from the gel is poor. Because there is. In addition, if the gel thickness is thicker than 0.75 mm, the moving distance when collecting the sample from the gel becomes large, and the sample may not be extracted and collected from the gel with high yield and high purity. Yes (see column (6) in the examples). In addition, the thickness of the gel of the sample insertion part 11 can be set suitably, and is not specifically limited.

  Further, a general commercially available gel preparation kit is designed so that a gel having a thickness of 1 mm can be prepared. For this reason, this number is a guideline for determining the gel thickness of the sample recovery part when preparing gel for electrophoresis using the attached gel plate (glass substrate) of such a commercially available gel preparation kit. Become.

  Further, since the gel thickness D1 of the sample loading unit 11 is larger than the gel thickness D2 of the sample collection unit 12, the sample can be loaded in the same manner as a conventional electrophoresis gel. For this reason, even when a dilute sample solution is used, a sufficient amount of sample can be introduced, and the analysis accuracy is improved. On the other hand, since the gel thickness D2 of the sample recovery unit 12 is thinner, even when the sample is extracted and recovered from the gel, the moving distance of the sample in the gel can be shortened.

  The “sample” is preferably a biological sample such as protein, peptide, DNA, RNA. As used herein, the “protein” may be a wild-type protein existing in nature or a mutant protein with a mutation, and is not particularly limited. Moreover, it can be used without any particular limitation, from a large molecular weight to a small molecular weight. “Peptide” is a substance formed by combining plural amino acids to several tens of amino acids in succession, and is a concept including so-called oligopeptides and polypeptides. Moreover, as shown in the Example mentioned later, after electrophoresis using protein as a sample, the peptide extracted and recovered by performing in-gel digestion is also included. Nucleic acids such as “DNA, RNA” may be double-stranded or single-stranded. The chain length is not particularly limited, and may be, for example, a primer composed of several bases or may consist of several thousand to several tens of thousands of base pairs. Further, for example, it may be a complex of nucleic acid and protein, or the protein may be post-translationally modified such as sugar chain modification.

  The gel for electrophoresis is used for extracting and collecting a sample separated by electrophoresis, but the specific method for “extracting and collecting” the sample from the gel here is limited. It is not something. For example, conventionally known means (method) using electrical or diffusion can be suitably used.

  The electrophoresis gel 10 may be any conventionally known electrophoresis gel used for analyzing a biological sample in the field of molecular biology, and its material, composition, concentration, and type of buffer solution. Specific conditions / configurations such as size and shape are not particularly limited. For example, it may be a polyacrylamide gel based gel capable of analyzing a relatively low molecular weight sample (eg, protein or peptide), or a gel such as an agarose gel for analyzing a relatively high molecular weight sample (eg, nucleic acid). Good.

  The electrophoresis gel may include a concentrated gel and a separation gel, and the sample recovery unit may be formed at least in part of the separation gel. A specific configuration of the electrophoresis gel in this case will be described with reference to FIG. FIG. 2 (a) is a diagram schematically showing a planar configuration of another electrophoresis gel according to the present embodiment, and FIG. 2 (b) shows the electrophoresis gel of FIG. It is a figure which shows typically the structure of the cross section at the time of cut | disconnecting by.

  As shown in FIG. 2A, the electrophoresis gel 20 according to the present embodiment includes a concentrated gel (stacking gel) 23 and a separation gel 24, and at least a part of the concentrated gel 23. A sample input unit 21 is provided, and a sample recovery unit 22 is provided on at least a part of the separation gel 24. Further, as shown in FIG. 2B, the thickness of the gel in the sample recovery unit 22 is formed thinner than the thickness of the gel in the sample input unit 21.

  According to the above configuration, the sample can be concentrated by the concentrated gel 23. For this reason, there is an advantage that the concentrated sample can be efficiently recovered by the sample recovery unit 22 formed in a part of the separation gel 24.

  Further, in the electrophoresis gel according to the present invention, the sample recovery unit only needs to be provided at least in a part of the separation gel. For example, as shown in FIGS. In the electrophoresis gel 30 including the separation gel 34, for example, a sample input part 31 is provided in at least a part of the concentrated gel 33, and a substantially entire surface of the separation gel 34 is formed as the sample recovery part 32. Also good. As shown in FIG. 3 (b), the gel thickness of the sample recovery unit 32 is formed thinner than that of the sample input unit 31.

  According to the above configuration, since the entire surface of the separation gel can be used as a region for sample collection, the sample can be collected even when the migration pattern of the sample cannot be accurately predicted, There is an advantage that the sample can be efficiently recovered at the same time.

  In addition, the electrophoresis gel according to the present embodiment, the electrophoresis gel is sandwiched and held between two substrates, and due to the difference in thickness between the sample input part and the sample recovery part, It is preferable that a space filling means is provided in a gap generated between the substrate and the sample recovery unit. A specific configuration of the electrophoresis gel in this case will be described with reference to FIG. As shown in the figure, the electrophoresis gel 40 is sandwiched and held by two glass substrates 46a and 46b. The electrophoresis gel 40 is provided with a sample input part 41 and a sample recovery part 42. Since the gel thickness of the sample collection unit 42 is formed to be thinner than the gel thickness of the sample insertion unit 41, when the sample insertion unit 41 and the two glass substrates 46a and 46b are in close contact with each other, the glass substrate 46b and the sample collection are collected. A space is generated between the portion 42. Spacers 45 functioning as space filling means are inserted in this space. The spacer 45 is in close contact with the glass substrate 45b.

  The gel for electrophoresis is usually used while being sandwiched between glass substrates. For this reason, if there is a space between the sample recovery part 42 and the glass substrate 46b, the gel becomes unstable. If the spacer 45 is used, such a problem can be avoided. Further, as will be described later, an electrophoresis gel is usually prepared by injecting a gel composition before solidification into a gap between two glass substrates. In this case, if a spacer 45 is used, the gel thickness is very simple. The thin sample collection part 42 can be produced.

  FIG. 4 illustrates the case where one glass substrate 46a and the sample recovery unit 42 are in close contact with each other. However, not only in such a case, for example, between the glass substrate 46a and the sample recovery unit 42. , And a spacer may be provided both between the glass substrate 46 b and the sample recovery part 42. In this case, the gel thickness of the sample recovery part can be appropriately set by adjusting the thicknesses of the two spacers.

  Further, as shown in FIG. 5, the spacer 45 is preferably a flat plate-like thin layer or a laminate of two or more flat plate-like thin layers. According to this configuration, the thickness of the sample recovery unit 42 can be easily changed by increasing or decreasing the number of stacked flat thin layers. The spacer 45 may be provided integrally with the glass substrate 46b, but is preferably provided separately on the glass substrate 46b. This is because the position, size, and thickness of the sample recovery unit can be arbitrarily changed by removing the spacer 45 and changing it appropriately. Furthermore, there is an advantage that the gel plate (glass substrate) attached to the commercially available gel preparation kit can be used as it is only by separately preparing the spacer 45.

  After electrophoresis using the electrophoresis gel having the spacer as described above, for example, when the gel is stained or cut out, the gel (carrier) is peeled off from the spacer after electrophoresis. Staining and sample collection. Further, for example, in the present invention, an adhesive is applied by appropriately selecting the material of the spacer (for example, a material having high adhesion to the gel, cellophane film, etc.) or between the spacer and the gel (carrier). In this case, the spacer and the gel can be in a fixed state (a state in which the spacer and the gel are in close contact with each other). Thus, when the gel and the spacer are integrally fixed, there is an advantage that the operability of the gel after electrophoresis is improved. That is, although the gel thickness of the sample collection part is thin, the thin gel part (sample collection part) is provided by providing a spacer as a support member (protective member) in this thin gel part. Can be avoided. For example, the risk of breaking or breaking the gel due to careless work is reduced. Note that it has been proved that there is no inconvenience in the operation of dyeing the gel or cutting out and collecting the sample from the gel even when the gel and the spacer are fixed.

  As described above, the gel for electrophoresis according to the present embodiment is formed so that the gel thickness of the sample recovery part is thinner than the gel thickness of the sample input part. That is, the volume of the gel in the sample recovery unit is smaller than that of a conventional electrophoresis gel. For this reason, when the sample (for example, protein etc.) isolate | separated by the sample collection | recovery part is extracted and collected, the movement distance of the sample at the time of moving a sample from the inside of a gel to the outside of a gel can be made small. Therefore, according to the gel for electrophoresis according to the present embodiment, the sample can be recovered with high yield from the gel piece containing the sample after electrophoresis.

[2] Method for Producing Electrophoresis Gel In the method for producing an electrophoretic gel according to the present invention, at least two substrates are arranged to face each other at a predetermined interval. A space filling means is provided in the gap provided between the substrates so as to be in contact with one of the substrates and not in contact with the other substrate, and the gel composition before solidification is provided in the gap between the two substrates. It is only necessary that the sample recovery unit is formed in the gap between the substrate not in contact with the space filling means and the space filling means by injecting an object, and other processes, conditions, materials, etc. are particularly limited. It is not something. Hereinafter, this manufacturing method will be described for each process based on the drawings.

  FIG. 6 is a drawing schematically showing a method for producing an electrophoresis gel according to the present embodiment. As shown in the figure, the method for manufacturing an electrophoresis gel according to the present embodiment includes two glass substrates 46a and 46b, a gap 50 that functions as a gap means, and a spacer 45 that functions as a space filling means. Use. The gap 50 is fixed to the glass substrate 46b. The material of the gap 50 and the spacer 45 is not particularly limited, but in this embodiment, the gap 50 is made of glass, and the spacer 45 is made of resin (polyethylene terephthalate).

  The two glass substrates 46a and 46b are moved in the direction indicated by the arrow B in the figure and arranged to face each other. At this time, a gap 50 is provided in one glass substrate 46b. For this reason, the two glass substrates 46a and 46b are arranged so as to face each other with a predetermined interval by overlapping the two glass substrates 46a and 46b via the gap 50. Become. That is, the two glass substrates 46a and 46b are arranged so as to face each other substantially in parallel with a predetermined interval.

  In addition, here, two gap portions 50 and 50 having a predetermined thickness are arranged at opposite ends of the glass substrate 46b so that the interval between the two glass substrates 46a and 46b is a predetermined interval. ing. In other words, the gap 50 is sandwiched between the two glass substrates 46a and 46b so that a predetermined space exists between the two glass substrates 46a and 46b. The gap 50 is provided in a region that is not a region for producing a gel between the two glass substrates 46a and 46b, for example, at both end portions between the two glass substrates 46a and 46b as in the present embodiment. It is preferable.

  In addition, as shown by an arrow A in FIG. 6, a gap provided between the two glass substrates 46a and 46b is in contact with one glass substrate 46b and not in contact with the other glass substrate 45a. A spacer 45 is provided.

  FIG. 7A is a schematic diagram showing a planar configuration when two glass substrates 46a and 46b are overlapped with a gap 50 and a spacer 45 is provided. FIG. It is sectional drawing which shows the cross section at the time of cut | disconnecting by the L4-L4 line in (a), FIG.7 (c) is sectional drawing which shows the cross section at the time of cut | disconnecting by the L5-L5 line in (a).

  As shown in FIGS. 7B and 7C, the spacer 45 is provided in a space (gap) that exists between the two glass substrates 46 a and 46 b overlapped with each other via the gap 50. The spacer 45 is in contact with one glass substrate 46b and is not in contact with the other glass substrate 45a. The spacer 45 is in close contact with the one glass substrate 46b. This is because when the spacer 45 and the glass substrate 46b are not in close contact, the gel before solidification flows into the gap and the gel may be produced in an unintended portion.

  Therefore, it is preferable that the spacer 45 has a flat plate shape so as to be in close contact with the one glass substrate 46b. Further, since the spacer 45 is in close contact with one glass substrate 46b and does not contact the other glass substrate 45a, the two glass substrates 46a and 46b and the spacer 45 are provided substantially in parallel. It will be.

  Then, by injecting the pre-solidification gel composition into the gap between the two glass substrates 46a and 46b, the sample recovery part is formed in the space 51 formed between the glass substrate 46a not in contact with the spacer 45 and the spacer 45. Will be. Since no gel is formed in the region where the spacer 45 is provided, the sample recovery unit is formed with a thinner gel than the sample input unit. The “pre-solidification gel composition” is a liquid gel composition before solidification.

  In addition, the spacer 45 is preferably a flat plate-like thin layer or a laminate of two or more flat plate-like thin layers, as shown in FIG. In this case, it is possible to arbitrarily change the thickness of the sample recovery unit by appropriately increasing or decreasing the number of stacked thin plate layers. Moreover, there is an advantage that the gel plate (glass substrate) attached to the commercially available gel preparation kit can be used as it is by appropriately changing the shape of the spacer 45. This is very useful in terms of cost because it is not necessary to produce a new gel plate (glass substrate) by using a widely used gel plate (glass substrate).

  Further, as described in the above section [1], for example, in the present invention, by appropriately selecting the material of the spacer (for example, a material having high adhesion to the gel), or the spacer and the gel (carrier) ), The gel for electrophoresis can be prepared by fixing the spacer and the gel. Thereby, the gel for electrophoresis which provided the spacer as a supporting member in the thin part (sample collection | recovery part) of gel thickness easily can be produced.

  In this embodiment, a glass substrate is used as the “substrate”. However, the present invention is not limited to this. For example, any conventionally known substrate used for manufacturing an electrophoresis gel may be used. Further, it is preferable that the two substrates have substantially the same size. A commercially available gel preparation kit can be used as the substrate and the gap.

  Thus, according to the method for producing an electrophoresis gel according to the present invention, an electrophoresis gel capable of efficiently collecting a sample can be produced very simply and easily. Further, by adjusting the structure such as the shape and thickness of the spacer (space filling means), a commercially available gel preparation kit can be used as it is, and there is a very excellent merit in terms of cost.

[3] Production Kit for Electrophoresis Gel As described above, a commercially available gel preparation kit can be used as it is by appropriately changing the structure of the spacer as the space filling means and using the electrophoresis according to the present invention. Gel can be prepared. For this reason, the manufacturing kit of the gel for electrophoresis provided with the space filling means at least is also contained in this invention. According to such a production kit, the electrophoresis gel according to the present invention can be easily produced, and the method for producing an electrophoresis gel according to the present invention can be easily carried out.

Moreover, it is preferable that the manufacturing kit further includes at least one of the following members (a) to (c).
(A) At least two substrates (b) Gap means for arranging two substrates to face each other with a predetermined gap (c) Gel material Here, “substrate” and “gap means” The above can be used as appropriate. The “gel material” is not particularly limited as long as it is a gel material used in a conventionally known gel for electrophoresis or a composition thereof, but a monomer, a crosslinking agent, a polymerization start is not limited. Agents, polymerization accelerators and the like. For example, when preparing a polyacrylamide gel for electrophoresis, acrylamide, N, N′-methylenebisacrylamide, ammonium persulfate, N, N, N ′, N′-tetramethylethylenediamine and the like can be mentioned. In addition to these gel materials, it may contain a buffer reagent, a pH adjuster (for example, tris (hydroxymethyl) aminomethane, hydrochloric acid, etc.), a surfactant (sodium dodecyl sulfate, etc.), etc. Good.

[4] Electrophoresis device As described above, the gel for electrophoresis according to the present invention has an advantage of excellent sample recovery efficiency. For this reason, it can be said that the electrophoresis apparatus which uses the said gel for electrophoresis as a support body also has the same effect. That is, the present invention includes such an electrophoresis apparatus.

  The electrophoretic device according to the present invention is not particularly limited as long as the electrophoretic gel is provided as a support, and other specific members and structures are not particularly limited. That is, in addition to providing the electrophoresis gel as a support, a member used in a conventionally known electrophoresis apparatus may be provided. For example, an electrophoretic tank and electrodes for performing electrophoresis by immersing the support in a buffer solution, a power source for applying voltage, and the like may be provided.

[5] Sample recovery method The sample recovery method according to the present invention is to perform electrophoresis using the electrophoresis gel according to the present invention, separate the sample, and extract and recover the sample from the sample recovery unit. Other specific processes, conditions, instruments, materials and the like are not particularly limited. As the method for extracting and recovering the sample, a conventionally known method can be suitably used as described above.

  According to this method, a sample separated for each molecular weight by electrophoresis can be recovered with high purity and efficiency, and can be used in various bio fields. The sample is preferably a nucleic acid such as a protein, peptide, DNA, or RNA.

  Hereinafter, examples will be shown, and the embodiment of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

(1) Preparation of reagent solution for acrylamide gel preparation A 30% acrylamide solution was prepared by dissolving 73 g of acrylamide (manufactured by Wako Pure Chemical Industries) and 2 g of N, N′-methylenebisacrylamide (manufactured by Wako Pure Chemical Industries) in distilled water. Obtained as 250 ml. 0.75M Tris (hydroxymethyl) aminomethane pH 8.8 solution was prepared by dissolving 90.75 g of tris (hydroxymethyl) aminomethane (manufactured by Nacalai Tesque) in 800 ml of distilled water and adjusting the pH to 8.8 with hydrochloric acid. Distilled water was added to obtain 1 l. A 1.0M tris (hydroxymethyl) aminomethane pH 6.8 solution was prepared by dissolving 121 g of tris (hydroxymethyl) aminomethane (manufactured by Nacalai Tesque) in 800 ml of distilled water, adjusting the pH to 6.8 with hydrochloric acid, and then adding distilled water. To obtain 1 liter. A 10% sodium lauryl sulfate solution was obtained as 100 ml by dissolving 10 g sodium lauryl sulfate (manufactured by Nacalai Tesque) in distilled water. A 10% ammonium persulfate solution was obtained as 1 ml by dissolving 100 mg ammonium persulfate (manufactured by Nacalai Tesque) in distilled water.

  The electrophoresis buffer was obtained as 1 liter by adding distilled water to 3 g of tris (hydroxymethyl) aminomethane (manufactured by Nacalai Tesque), 14.4 g of glycine (manufactured by Wako Pure Chemical Industries) and 1 g of sodium lauryl sulfate. The sample addition solution was 0.5 ml of 1.0 M tris (hydroxymethyl) aminomethane pH 6.8 solution, 0.154 g of dithiothreitol (manufactured by Nacalai Tesque), 2 ml of 10% sodium lauryl sulfate solution, bloom phenol blue (Wako Pure Chemical Industries, Ltd.) Distilled water was added to 0.01 g of glycerol (manufactured by Nacalai Tesque) and 1 ml to obtain 10 ml.

(2) Preparation of conventional acrylamide gel (comparative example)
A gel was prepared using a mini slab gel preparation kit (AE6401 manufactured by Ato). Assemble the gel plate and use 2ml of 30% acrylamide solution, 5.3ml of distilled water, 2.5ml of 0.75M tris (hydroxymethyl) aminomethane pH8.8 solution, 0.1ml of 10% sodium lauryl sulfate solution, After mixing 0.1 ml of 10% ammonium persulfate solution, the solution added with 0.008 ml of N, N, N ′, N′-tetramethylethylenediamine was poured into the gel plate, and distilled water was layered on top of the liquid. Gelation was performed.

  After the gelation, the upper distilled water was discarded and a comb was set. Next, as a stacking gel, 1.4 ml of distilled water, 0.33 ml of 30% acrylamide solution, 0.25 ml of 1.0M tris (hydroxymethyl) aminomethane pH6.8 solution, 0.02 ml of 10% sodium lauryl sulfate solution, After mixing 0.02 ml of 10% ammonium persulfate, a solution to which 0.002 ml of N, N, N ′, N′-tetramethylethylenediamine was added was poured and solidified.

(3) Preparation of acrylamide gel according to the present embodiment Using a mini slab gel preparation kit (AETO AE6401), as shown in FIG. 8, a 0.5 mm thick polyethylene terephthalate plate (space filling) as shown in FIG. The gel plate was assembled by placing it on a glass substrate on one side. As a gel for separation, 2 ml of 30% acrylamide solution, 5.3 ml of distilled water, 2.5 ml of 0.75M tris (hydroxymethyl) aminomethane pH 8.8 solution, 0.1 ml of 10% sodium lauryl sulfate solution, 10% ammonium persulfate solution After mixing 0.1 ml, the solution added with 0.008 ml of N, N, N ′, N′-tetramethylethylenediamine is poured into a gel plate, and distilled water is overlaid on top of the liquid to perform gelation. It was.

  After the gelation, the upper distilled water was discarded and a comb was set. Next, as a stacking gel, 1.4 ml of distilled water, 0.33 ml of 30% acrylamide solution, 0.25 ml of 1.0M tris (hydroxymethyl) aminomethane pH6.8 solution, 0.02 ml of 10% sodium lauryl sulfate solution, After mixing 0.02 ml of 10% ammonium persulfate, a solution to which 0.002 ml of N, N, N ′, N′-tetramethylethylenediamine was added was poured and solidified.

(4) Electrophoretic analysis After gelation, the comb was extracted and set in a mini slab electrophoresis tank (manufactured by ATTO), and the upper and lower ends of the gel were immersed in an electrophoresis buffer. As protein samples, 2 mg / ml of protein standard for electrophoresis (manufactured by Bio-Rad) is diluted 1000 times with a sample addition solution, and each protein is contained in 4 types including 1.5 ng, 5 ng, 10 ng, and 20 ng in 10 μl. A sample solution was prepared and the whole amount was put into a well. Thereafter, a current of 20 mA (milliampere) was applied to perform electrophoresis. When the dye marker reached the lower end of the gel, the electrophoresis was terminated, the gel plate was disassembled, the gel was taken out, and stained with a silver staining MS kit (manufactured by Wako Pure Chemical Industries).

  A band corresponding to phosphorylase (shown as “97.4k” in FIG. 9) was cut out from a 10 ng protein input lane, digested in gel, followed by mass spectrometry, and an attempt was made to identify the peptide obtained from phosphorylase. The operation from in-gel digestion to mass spectrometry was carried out by the method of Shevchenko et al. Described in Analytical Chemistry 68, 850-858, 1996.

(5) Results Similar to the conventional acrylamide gel (1 mm thickness) shown in the above (2) column, the acrylamide gel according to the present embodiment shown in the (3) column (the sample recovery part is 0.5 mm thick). Other parts were 1 mm thick).

  Moreover, as a result of electrophoresis, it shows to Fig.9 (a) (b). In the figure, the numerical values (1.5n, 5n, 10n, and 20n) at the top of the gel indicate the amount of protein in each loaded sample. As shown in FIGS. 9 (a) and 9 (b), the gel with the sample collection part having a thickness of 0.5 mm has a clear separated protein band as compared with the conventional gel having a thickness of 1 mm. It was. That is, it can be said that the gel whose sample recovery part has a thickness of 0.5 mm has higher resolution than the conventional one.

  In addition, FIG. 10 shows the results of mass spectrometry analysis of peptides extracted and recovered from the gel after electrophoresis. 10A and 10B, the vertical axis indicates the peak intensity.

  As shown in FIGS. 10 (a) and 10 (b), the signal intensity of a peptide derived from phosphorylase extracted from a gel having a thickness of 0.5 mm in the sample recovery part (indicated by an arrow in the figure) Was about 5 times the peptide extracted from a 1 mm thick gel. Furthermore, the number of peptides that can be detected through noise is 22 in the gel whose sample collection part is 0.5 mm thick, compared with 7 in the conventional gel having a thickness of 1 mm. . This is a number more than three times. As a result of analysis using 10 ng of protein as a sample, the accuracy of protein identification in database search based on peptide mass obtained by mass spectrum was improved (effective peptide identification rate: 15% → 47%, protein sequence coverage: 9% → 31%).

  From the above, it was found that the electrophoresis gel according to the present embodiment is significantly more efficient in recovering proteins and peptides from the gel than the conventional gel.

(6) Sample recovery comparative experiment from gel according to this embodiment and conventional gel Further, a gel for electrophoresis in which the gel thickness of the sample recovery part was 0.75 mm was prepared by the same method as described above. Using this electrophoresis gel with a sample collection part of 0.75 mm and a conventional electrophoresis gel with a total thickness of 1.0 mm, the peptide digested in the gel after electrophoresis is collected, The recovery efficiency was compared.

  Specifically, after the sample recovery unit separates the protein sample by electrophoresis using a gel having a thickness of 0.75 mm and a gel having a thickness of 1 mm, the sample protein is enzymatically digested in the gel, and the generated peptides are respectively The mass spectrum was collected from the gel.

  The result is shown in FIG. FIG. 11 (a) is a diagram showing a mass spectrum of a peptide recovered from a gel with a gel thickness of 0.75 mm in the sample recovery part, and FIG. 11 (b) is recovered from a gel with a gel thickness of 1mm in the sample recovery part. It is a figure which shows the mass spectrum of a peptide. As shown in the figure, it was found that the baseline noise was higher in the gel having a gel thickness of 1.0 mm than in the gel having a gel thickness of 0.75 mm in the sample recovery portion. This is because many impurities contained in the gel are mixed. That is, it was found that when the gel thickness of the sample recovery part was 0.75 mm, the sample could be recovered with high purity and high yield compared to the conventional gel having a thickness of 1.0 mm. Although an attempt was made to create a thin electrophoresis gel having a gel thickness of less than 0.1 mm in the sample collection part, there was a case where a uniform quality gel could not be created.

  From the above, it can be said that the gel thickness of the sample recovery part is preferably 0.1 mm or more and 0.75 mm or less.

(7) Gel fixed to sheet (spacer) Acrylamide gel was prepared as shown in the above-mentioned columns (1) to (3) in principle. Specifically, a mini slab gel preparation kit (AE6401 manufactured by ATTO) was used. A gel plate was assembled by adhering a cellophane membrane (Wako Pure Chemicals “Dialysis Membrane”) damped with water to one of the two glass plates that create a space for creating a gel. . That is, a cellophane film is used as the spacer. For separation gel, 2 ml of 30% acrylamide solution, 5.3 ml of distilled water, 0.75 M and 2.5 ml of Lith <hydroxymethyl> aminomethane pH 8.8 solution, 0.1 ml of 10% sodium lauryl sulfate solution, 10% After mixing 0.1 ml of ammonium persulfate solution, a solution containing 0.008 ml of N, N, N ′, N′tetramethylethylenediamine was poured into the gel plate, and distilled water was layered on top of the liquid to gelate. Went.

  After gelation, the upper distilled water was discarded and a comb was set. Next, as a stacking gel, 1.4 ml of distilled water, 0.33 ml of 30% acrylamide solution, 0.25 ml of 1.0M and Lith <hydroxymethyl> aminomethane pH 6.8 solution, 0.02 ml of 10% ammonium persulfate solution, After mixing 0.02 ml of 10% ammonium persulfate solution, a solution added with 0.002 ml of N, N, N ′, N′tetramethylethylenediamine was poured and solidified.

  After gelation, the comb was extracted and set in a mini slab electrophoresis tank (manufactured by ATTO), and the upper and lower ends of the gel were immersed in an electrophoresis buffer.

  As a protein sample, 100 ng of human serum albumin and 5 μl of prestained SDS-PAGE standard (manufactured by Biorad) were added to the well, and then 20 mA of current was applied for electrophoresis. When the dye marker reached the lower end of the gel, the electrophoresis was terminated, the gel plate was disassembled, the gel was taken out, and stained with a silver staining MS kit (manufactured by Wako Pure Chemical Industries).

  As a result, as shown in FIG. 12, the gel was not peeled off from the cellophane film in the staining operation after electrophoresis, and the handling and operability were very good. There was no effect on staining.

  As described above, the gel for electrophoresis according to the present invention can collect a gel electrophoresis fraction of a sample (for example, protein, peptide, nucleic acid, etc.) with high purity and high efficiency. Very useful for recovery. In other words, it can be used not only for academic utility but also for a very wide range of applications such as research and development for various bio-industries (food, medicine, environment, etc.) and quality control of protein products.

(A) is a figure which shows typically the structure of the plane of the gel for electrophoresis which concerns on this Embodiment, (b) is the case where the gel for electrophoresis of (a) is cut | disconnected by the L1-L1 line | wire. It is a figure which shows the structure of a cross section typically, (c) is a figure which shows typically the other structure of a cross section at the time of cut | disconnecting the gel for electrophoresis of (a) by the L1-L1 line. (A) is a figure which shows typically the structure of the plane of the other electrophoresis gel which concerns on this Embodiment, (b) cut | disconnected the gel for electrophoresis of (a) by the L2-L2 line | wire. It is a figure which shows typically the structure of the cross section in a case. (A) is a figure which shows typically the structure of the plane of the other gel for electrophoresis which concerns on this Embodiment, (b) cut | disconnected the gel for electrophoresis of (a) by the L3-L3 line | wire. It is a figure which shows typically the structure of the cross section in a case. It is sectional drawing which shows typically the structure of the cross section of the other gel for electrophoresis which concerns on this Embodiment. It is a figure which shows typically the structure of the spacer which concerns on this Embodiment. It is drawing which shows typically the manufacturing method of the gel for electrophoresis which concerns on this Embodiment. (A) is the schematic diagram which shows the structure of a plane at the time of overlapping two glass substrates through a gap | interval and providing a spacer, (b) is the L4-L4 line | wire in (a). It is sectional drawing which shows the cross section at the time of cut | disconnecting by (c), (c) is sectional drawing which shows the cross section at the time of cut | disconnecting by the L5-L5 line | wire in (a). It is a figure which shows the gel for electrophoresis in a present Example. (A) is a figure which shows the result after electrophoresis of the conventional gel for electrophoresis, (b) is a figure which shows the result after electrophoresis with the gel for electrophoresis which concerns on a present Example. (A) is a figure which shows the result of having carried out mass spectrometry of the peptide extracted and collected after in-gel digestion from the gel for electrophoresis which concerns on a present Example, (b) is extracted and recovered after in-gel digestion from the gel for conventional electrophoresis. It is a figure which shows the result of having conducted mass spectrometry of the obtained peptide. (A) is a figure which shows the mass spectrum of the peptide collect | recovered from the gel whose gel thickness of a sample collection part is 0.75 mm, (b) is the mass spectrum of the peptide collect | recovered from the gel whose gel thickness of a sample collection part is 1 mm FIG. In a present Example, after performing electrophoresis using the gel for electrophoresis in which the spacer (cellophane film | membrane) and gel (sample collection part) contact | adhered, it is a figure which shows the result at the time of dyeing | staining process.

Explanation of symbols

10, 20, 30, 40 Electrophoresis gel 11, 21, 31, 41 Sample input part 12, 22, 32, 42 Sample recovery part 23, 33 Concentrated gel 24, 34 Separation gel 45 Spacer (space filling means)
46a, 46b Glass substrate (substrate)
50 Gap (gap means)

Claims (14)

An electrophoresis gel used for extracting and recovering a sample separated by electrophoresis from a gel,
A sample input part for inputting the sample and a sample recovery part for recovering the separated sample are formed,
The gel for electrophoresis characterized in that the thickness of the gel in the sample recovery part is formed thinner than the thickness of the gel in the sample input part. The electrophoresis gel includes a concentrated gel and a separation gel,
The gel for electrophoresis according to claim 1, wherein the sample recovery part is formed at least in a part of the separation gel.   The electrophoresis gel according to claim 1 or 2, wherein the thickness of the sample recovery part is 0.1 mm or more and 0.75 mm or less. The electrophoresis gel is sandwiched and held between two substrates,
4. A space filling means is provided in a gap generated between the substrate and the sample recovery part due to a difference in thickness between the sample input part and the sample recovery part. 2. The gel for electrophoresis according to item 1.   The gel for electrophoresis according to claim 4, wherein the space filling means is a thin plate layer or a laminate of two or more thin plate layers.   The gel for electrophoresis according to claim 4 or 5, wherein the space filling means and the sample recovery part are fixed to each other.   The gel for electrophoresis according to any one of claims 1 to 6, wherein the sample is protein, peptide, DNA, or RNA. It is a manufacturing method of the gel for electrophoresis according to any one of claims 1 to 7,
At least two substrates are arranged to face each other with a predetermined interval,
A space filling means is provided in the gap provided between the two substrates so as to be in contact with one of the substrates and not in contact with the other substrate,
By injecting the gel composition before solidification into the gap between the two substrates, the sample recovery part is formed in the gap between the substrate not in contact with the space filling means and the space filling means. A method for producing a gel for electrophoresis.   9. The method for producing an electrophoresis gel according to claim 8, wherein the space filling means is a flat thin layer, or two or more flat thin layers are laminated.   An electrophoresis gel manufactured by the method for manufacturing an electrophoresis gel according to claim 8 or 9. A production kit for producing the gel for electrophoresis according to any one of claims 1 to 7 and 10,
A production kit comprising at least space filling means. Furthermore, it has at least 1 among the members of the following (a)-(c), The manufacturing kit of Claim 11 characterized by the above-mentioned.
(A) At least two substrates (b) Gap means for arranging two substrates so as to face each other with a predetermined gap (c) Gel material   An electrophoresis apparatus comprising the electrophoresis gel according to any one of claims 1 to 7 and 10 as a support.   Electrophoresis using the gel for electrophoresis according to any one of claims 1 to 7 and 10, separating the sample, and then extracting and recovering the sample from the sample recovery unit .

Images