JP4822350B2 - Method of introducing sample into drying medium for electrophoresis and instrument for the same - Google Patents

Description

  The present invention relates to a method for introducing a sample into a drying medium for electrophoresis and a device therefor.

  Currently, two-dimensional electrophoresis (2DE) is a technique that is often used for protein analysis. Comparison of expression levels and analysis of post-translational modifications are important for analyzing protein functions. Since 2DE can simultaneously detect the amount of protein expression and post-translational modification, it will be used in clinical laboratories in the future. It is expected that it will be. Currently, 2DE that is widely used includes, for example, isoelectric focusing using a pH gradient gel in the first dimension and SDS-PAGE in the second dimension. The pH gradient gel used for the first-dimensional isoelectric focusing is a long and narrow strip-like gel, and a dry immobilized pH gradient gel (IPG) in which a gel is bound to a plastic film is used.

  Conventionally, a protein sample is introduced into an elongated strip-shaped IPG by immersing the IPG in a sample-containing swelling solution obtained by dissolving a protein sample in a gel swelling solution. That is, using a device having a groove wider than IPG, the sample-containing swelling liquid is placed in the groove, and IPG is immersed therein. The gel should be left free of dry portions and is therefore usually immersed overnight in a sufficient amount of sample-containing swelling solution to swell the entire gel.

  However, in this conventional method, even if it is immersed overnight, the reproducibility of the result of two-dimensional electrophoresis is not always satisfactory. That is, even if the same sample is subjected to two-dimensional electrophoresis using the same commercially available gel and the same method, different results are often obtained.

  Patent Document 1 describes a method for reducing the time required for introducing a sample by applying a voltage when the sample is introduced into the IPG. However, the method described in Patent Document 1 does not solve the problem of reproducibility, and it is considered that the time required for introducing the sample cannot necessarily be shortened depending on the charge of the sample protein.

JP 2005-345334 A JP 2006-258685

  The object of the present invention is to improve the reproducibility of the result of electrophoresis and to rapidly introduce the sample into the dry gel, and a method for introducing the sample into the drying medium for electrophoresis and an apparatus therefor Is to provide.

  The inventors of the present application have studied the cause that the reproducibility is not high by the conventional method, and as a result, even if the size of the commercially available dried gel is the same product, there is variation, and the sample-containing swelling liquid swells the gel The amount of protein sample introduced into the gel varies from gel to gel because the entire amount is not absorbed by the gel, but a part of it remains. I thought. As a result of research to solve this, the introduction of the sample into the dried gel and the swelling of the gel are divided into two stages. First, a small amount of sample solution is absorbed from the entire length of the gel in the longitudinal direction. Next, we sought to improve the reproducibility of two-dimensional electrophoresis by swelling the entire gel with the swelling solution and to shorten the sample introduction time, and confirm this experimentally. The present invention has been completed.

  That is, the present invention is a method of introducing a sample solution containing a sample to be subjected to electrophoresis into an elongated strip-shaped electrophoresis drying medium, and at least a part of a sample introduction surface that is one surface of the drying medium. The sample introduction region is a region that extends over substantially the entire length in the longitudinal direction of the sample introduction surface, and the sample solution is the dried solution. Less than the amount by which the entire medium can be swollen, and the drying medium is partially swollen by absorbing substantially the entire amount of the sample solution into the drying medium, and then the drying medium is swollen with the swelling liquid. A method for introducing a sample into an electrophoretic drying medium is provided. Further, the present invention is an instrument for performing the method of the present invention, for introducing a sample into an electrophoretic drying medium comprising an instrument body and the sample introduction groove provided in the instrument body. Providing equipment.

  The method of the present invention improves the reproducibility of electrophoresis. Further, in the method of the present invention, a small amount of the sample solution is absorbed from a region over the substantially entire length in the longitudinal direction of the drying medium, so that the time required for introducing the sample is shortened.

  The electrophoresis drying medium used in the method of the present invention is usually an electrophoresis drying gel, and is preferably a first-dimensional drying gel for two-dimensional electrophoresis, such as IPG. The drying medium has an elongated strip shape, and generally has a width of about 0.3 mm to 3 mm, a length of about 40 mm to 200 mm, and a thickness of about 0.05 mm to 0.2 mm. In addition, the electrophoretic dry gel is usually used in a form bound on a plastic film. In the present invention, a dry medium bound on such a plastic film can be preferably used. . Various dry gels for electrophoresis such as IPG bonded on a plastic film are commercially available. The commercially available products can be used as they are or after being cut into a desired size.

  The electrophoretic drying medium can be used as it is in the method of the present invention, but preferably, one surface thereof is directly or indirectly supported on a rigid support. By supporting the drying medium on a solid support, it is possible to prevent the drying medium from being distorted in the contact step with the sample solution described later, and the handleability is also improved. Furthermore, in the step of contacting with the sample solution, when the drying medium is immersed in a sample introduction groove composed of a narrow groove, which will be described later, a constant pressure can always be applied by its own weight. The drying medium may be supported directly on the support, or may be indirectly supported via the plastic film when the drying medium is bonded onto the plastic film as described above. As described above, since the drying medium is usually in a form bonded on a plastic film, the latter is preferable. The surface of the support that supports the drying medium has the same dimensions as the drying medium or is larger than the drying medium and supports the entire surface of the drying medium to prevent distortion of the drying medium. This is preferable. The thickness of the support is not particularly limited, but is preferably a thickness that can be easily pinched with a finger, and is preferably about 0.3 mm to 10 mm. The material of the support is not particularly limited, and plastic, ceramics, glass, or the like can be used, and it is advantageous from the viewpoint of ease of manufacture and price that it is made of plastic. That is, in a preferred embodiment, the support is in the form of a square bar-shaped plastic block.

  In the method of the present invention, the above-mentioned drying medium is first brought into contact with a sample solution having a smaller amount than the amount capable of swelling the entire drying medium, so that substantially the entire amount is absorbed by the drying medium, and thereafter Then, the drying medium is brought into contact with the swelling liquid, and the whole is swollen so that no dried portion remains. Here, “substantially the entire amount” means the entire amount or almost the entire amount, and the degree of wetness of the surface of the groove for storing the sample solution (sample introduction groove to be described later) is good, but it is a liquid having a liquid surface. This means that it does not remain in this state. The sample may be any substance to be analyzed by electrophoresis, and is often a protein, peptide, nucleic acid or the like, but is not limited thereto. The solvent of the sample solution is not particularly limited, but is usually an aqueous buffer solution, and a gel swelling solution may be used as the solvent. As the gel swelling solution, usually, for example, 8M urea, 2% 3- [3- (chloroamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), 50 mM dithiothreitol, and 0.2% amphoteric electrolyte. Aqueous solutions containing water are used, and these are attached to commercially available two-dimensional electrophoresis kits.

  The sample solution is introduced into the sample introduction region by bringing the sample introduction region, which is at least a part of the sample introduction surface, which is one surface of the drying medium, into direct contact with the sample solution. The sample introduction region is a region extending over substantially the entire length in the longitudinal direction of the drying medium. Here, “substantially full length” means the full length or almost the full length, and the areas about several millimeters from both ends, such as the area for connecting the electrodes to both ends, are not in contact with the sample solution. Also good.

Hereinafter, a preferred specific example of the sample introduction method will be described with reference to the drawings. FIG. 1 illustrates a method using an instrument particularly suitable for sample introduction. The instrument 10 includes an instrument body 12 having an elongated rectangular parallelepiped shape, and a groove 14 is formed on the upper surface of the instrument body 12. As will be described later, the groove 14 serves as a guide for the drying medium supported by the support, and hence is referred to as a “guide groove”. A narrower groove 16 is formed on the bottom surface of the guide groove 14, and this thin groove 16 accommodates a sample solution and introduces the sample solution into the drying medium, and is therefore referred to as a “sample introduction groove”. Although the material which comprises the instrument 10 is not specifically limited, Plastic is normally used from the point of the ease of shaping | molding and a price. In FIG. 1, reference numeral 18 denotes a dry gel bonded on a plastic film, and 20 denotes a support made of a square-bar plastic block that supports the dry gel 18 through the plastic film. In FIG. 1, two supports 20 supporting the dry gel 18 with plastic film are drawn. This is because the dry gel 18 with plastic film 18 and the sample solution before contact with the sample solution are shown. In order to show the dried gel 18 with plastic film in a state after contact with the sample film, the dried gel 18 with plastic film before and after the contact with the sample solution is drawn respectively. There is only one.

FIG. 2 schematically shows a cross-sectional view of the instrument 10 shown in FIG. 1 cut in a direction perpendicular to the longitudinal direction. Note that FIG. 2 is a schematic diagram for explaining the instrument and how to use it, and the dimensional ratio is not necessarily accurate. As well shown in FIG. 2, a guide groove 14 is formed on the upper surface of the instrument body 12, and a sample introduction groove 16 is formed on the bottom thereof. In FIG. 2, reference numeral 18a is a dry gel, 18b is a plastic film to which the dry gel 18a is bonded, and in the illustrated example, the dry gel 18a, the plastic film 18b, and the support 20 are planes viewed from above. The same dimensions and shape. The width of the guide groove 14 is slightly larger (usually about 0.2 mm to 1.0 mm) than the width of the support 20. The depth of the guide groove 14 is determined when the dry gel 18 with plastic film and the support 20 (hereinafter referred to as “support with dry gel” for convenience) are placed on the bottom surface of the guide groove 14. It is preferable that the height of the support with dry gel is shallower than the height of the support with dry gel so that the top of the support protrudes from the guide groove 14 to such an extent that it can be pinched with a finger. The length of the guide groove 14 only needs to be longer than the length of the support with dry gel, and is usually about 1 mm to several mm longer than the length of the support with dry gel. The width of the sample introduction groove 16 is not particularly limited, but is about 20% to 80% of the width of the dry gel, and is about 1/3 in the illustrated example. The position where the sample introduction groove 16 is provided is not particularly limited, but is preferably near the center in the direction perpendicular to the longitudinal direction of the bottom surface of the sample introduction groove 16 as shown in FIG. Further, the depth of the sample introduction groove 16 is such that the sample solution is filled up to the upper end of the sample introduction groove 16, and the entire dry gel 18a is swollen even when the entire amount of the sample solution is absorbed by the dry gel 18a. Although it is the depth which becomes the amount which cannot be performed and it does not specifically limit, About 2 to 4 times the thickness of the dry gel 18a is preferable normally. In the example shown in FIG. 2, the angle between the bottom surface and the side surface of the guide groove 14 is depicted as 90 degrees, but this portion may be rounded, and the bottom of the guide groove 14 is shaped like a mortar. It may be. Similarly, the angle between the bottom surface and the side surface of the sample introduction groove 16 is also drawn at 90 degrees. However, this portion may be rounded, and the cross-sectional shape of the sample introduction groove 16 is not limited to a rectangle but is half. It may be circular or semi-elliptical.

  When introducing a sample into the dry gel 18a, first, the sample solution is put into the sample introduction groove 16. It is preferable to put the sample solution up to the edge of the upper end of the sample introduction groove 16. By doing so, the liquid level of the sample solution is slightly lifted in a bow shape from the bottom surface of the guide groove 14 due to surface tension. In this state, the support with dry gel is inserted into the guide groove 14 (downward arrow in FIG. 2) and placed on the bottom surface of the guide groove 14. At this time, since the width of the guide groove 14 is only slightly wider than the width of the support body 20, the support body 20 is guided by the guide groove 14. In particular, when the lower portion of the guide groove 14 has a mortar shape, the guide groove 14 is more accurately guided to a fixed position. When a support with a dry gel is placed on the bottom surface of the guide groove 14, the region of the dry gel 18a that contacts the opening of the sample introduction groove 16 (the “sample introduction region” in the present invention) is slightly affected by the surface tension. The raised sample solution is in direct contact with the sample solution accommodated in the sample introduction groove 16, and the sample solution is absorbed by the dry gel 18a, so that the sample introduction region of the dry gel 18a swells. Thus, the sample introduction groove 16 has a two-dimensional shape (planar shape seen from above) having substantially the same size and shape as the sample introduction region. Here, “substantially the same size and shape” means completely the same or almost the same size and shape. When the dried gel comes into contact with the sample solution, a small amount of the sample solution is at the upper end of the sample introduction groove 16. A difference that allows the width of the sample introduction region to be wider than the sample introduction groove 16 by leaking from the portion to the side is allowed. As will be described later, in the case where the bottom surface of the sample introduction groove 16 is subjected to a hydrophilic treatment, the sample solution does not spread to a portion having a hydrophobic surface other than the portion subjected to the hydrophilic treatment. It is considered that the sample introduction groove 16 is constituted only by the region subjected to the crystallization treatment. That is, in this case, the region subjected to the hydrophilic treatment has substantially the same size and shape as the sample introduction region. When the support with the dried gel is placed on the bottom surface of the guide groove 14, if the same support is used due to the weight of the support 20, the same pressure is applied to the dried gel 18a, and the sample introduction conditions are always constant. It ’s useful. When the dried gel 18a comes into contact with the sample solution in this way, the dried gel 18a is bonded to the plastic film 18b, and therefore hardly swells in the horizontal direction (horizontal direction) in FIG. The swollen gel protrudes into the sample introduction groove 16. This is schematically illustrated in FIG. That is, in FIG. 1, the state described as “IPG gel before swelling” is the state before contact with the sample solution, and the state described as “IPG gel after swelling” is the state after contact with the sample solution. Show. The curved arrows in FIG. 1 indicate the operation of taking out the support with dry gel after inserting it into the guide groove 14. As schematically shown in FIG. 1, in the dried gel after contact with the sample solution, the portion in contact with the opening of the sample introduction groove 16, that is, the sample introduction region in contact with the sample solution swells in the vertical direction. The contact time between the dried gel 18a and the sample solution is not particularly limited, but is usually about 1 minute to 20 minutes, particularly about 2 minutes to 10 minutes. The sample introduction is usually performed at room temperature.

  After the gel has absorbed substantially the entire amount of the sample solution, the support with gel is removed from the guide groove 14, and then the gel is immersed in a swelling solution to completely swell. In the method of the present invention, the swelling liquid used after the sample introduction does not include a valuable sample and is a relatively inexpensive one that is commercially available. Therefore, the swelling liquid is usually swollen in a groove-shaped container wider than the guide groove 14 described above. It is carried out by filling a sufficient amount of the liquid and immersing the support with gel in it.

  The gel swollen with the swelling liquid is then subjected to two-dimensional electrophoresis. The method of two-dimensional electrophoresis may be exactly the same as the conventional method. After applying isoelectric focusing by applying voltage in the one-dimensional direction (longitudinal direction of the gel), contact with the flat polyacrylamide gel containing SDS. In this state, the second-dimensional electrophoresis can be performed by applying a voltage in a two-dimensional direction (a direction perpendicular to the one-dimensional direction).

  As described above, the width of the dried gel is usually about 1 mm to 3 mm, and the width of the sample introduction groove is usually about 20% to 80%. Therefore, the width of the sample introduction groove is usually about 0.2 mm to 2.4 mm. It is a thin thing. In order to perform sample introduction in a short time with good reproducibility, the sample solution needs to be uniformly absorbed from the entire surface of the sample introduction region described above. For this reason, the sample solution uniformly spreads over the entire sample introduction groove. Need to be. As described above, since the sample introduction groove is thin, in order to spread the sample solution uniformly over the entire sample introduction groove, substantially the entire bottom surface of the sample introduction groove must be hydrophilized. preferable. If the entire surface of the bottom surface of the sample introduction groove is hydrophilized, if the sample solution is dropped onto a part of the sample introduction groove, the sample solution is uniformly distributed over the entire sample introduction groove. Yes, it is preferable. Here, “substantially the entire surface” means the entire surface or almost the entire surface, and even if there is a slight portion that has not been subjected to the hydrophilic treatment due to unevenness of the hydrophilic treatment, the sample solution Is uniformly distributed, it is included in the fact that “substantially the entire surface” has been subjected to the hydrophilic treatment.

The hydrophilization treatment can be performed, for example, by coating a hydrophilic polymer. The method of hydrophilization treatment is not particularly limited, but the hydrophilic coating layer preferably has a uniform thickness in order to spread the sample solution uniformly over the entire sample introduction groove, and the width of the sample introduction groove. Since it is narrow, it is not easy to apply the hydrophilic polymer with various known coaters. However, the polymer is polymerized in situ using plasma polymerization, and the bottom of the narrow groove is coated with a thin hydrophilic polymer layer with a uniform layer thickness by further processing with oxygen plasma or the like as necessary. (See Patent Document 2). Monomers that can be used for plasma polymerization include monomers having a hydrophilic main chain such as ether, and monomers having a hydrophilic group such as a hydroxyl group, amino group, and carboxyl group in the main chain or side chain, The polymer is not particularly limited as long as it can be polymerized by plasma polymerization. Examples of preferable monomers include oxygen-containing organic compounds such as acrylic acid and propargyl alcohol, acetonitrile, aminoacetaldehyde dimethyl acetal, propylamine, allylamine, Examples include nitrogen-containing organic compounds such as pyridine, but are not limited thereto. Alternatively, hydrophilic treatment may be performed by plasma treatment after plasma polymerization of a hydrophobic layer such as hexamethyldisiloxane. Furthermore, hydrophilization may be performed by simultaneously supplying hexamethyldisiloxane and oxygen and plasma-polymerizing to coat a silicon oxide film. This silicon oxide film can be formed by various thin film forming means other than the plasma polymerization method, for example, vapor deposition, sputtering, CVD, or the like. The hydrophilic layer can also be formed by treating the bottom surface of the sample introduction groove with oxygen plasma or the like. That is, the hydrophilic layer is preferably
(A) treatment with at least one plasma selected from the group consisting of hydrogen plasma, oxygen plasma and nitrogen plasma;
(B) The coating layer formed by plasma polymerization of at least one selected from the group consisting of hexamethyldisiloxane and hexadiene is subjected to the plasma treatment of (a) above, or (c) acrylic acid, propargyl alcohol, acetonitrile, It can be formed by plasma polymerizing at least one selected from the group consisting of aminoacetaldehyde dimethyl acetal, propylamine, allylamine, and pyridine.
(D) It can be formed by coating a silicon oxide film.

  The layer thickness of the hydrophilic layer is not particularly limited, but is preferably about 50 nm to 200 nm. The plasma polymerization and plasma processing techniques are well known, and devices for this are also commercially available, and can be easily implemented using commercially available devices (Patent Document 2). The sample introduction groove only needs to have at least a hydrophilic treatment on the bottom surface. Although the side surface may also be hydrophilized, the amount of the sample solution adsorbed in the sample introduction groove can be reduced by hydrophilizing only the bottom surface, so that the reproducibility can be further improved, which is preferable. .

  The length of the sample introduction groove may be the same as the length of the dry gel, or both ends may be shorter than each by several millimeters or less. In these cases, the support with the dry gel is attached to the guide groove. When placed on the bottom surface, there is a risk of bubbles entering between the sample solution and the dried gel. Therefore, by making the length of the sample introduction groove slightly longer than that of the dried gel, when the support with the dried gel is placed on the bottom surface of the guide groove 14, air holes are respectively formed outside the both ends of the dried gel. May be formed. In this case, if the sample introduction groove is too long than the dry gel, the amount of the required sample solution is increased, and substantially the entire amount of the sample solution may not be absorbed by the dry gel. The length of the sample introduction groove is small air of several mm or less, preferably about 0.2 mm to 1 mm in length outside the ends of the dry gel when the dry gel support is placed on the bottom surface of the guide groove. The length is preferably such that the holes are formed. In addition, the bottom surface of the part that becomes the air hole may be subjected to a hydrophilic treatment, but this part is not subjected to the hydrophilic treatment and is made a hydrophobic region so that the sample solution does not reach this part. (In this case, the length of the sample introduction groove is interpreted as the length of the portion subjected to the hydrophilization treatment as described above). In this way, the purpose of exhausting the bubbles is achieved, and it is not necessary to increase the amount of the sample solution.

  The above-described method using the instrument shown in FIG. 1 and FIG. 2 is a particularly preferable method, but the method of the present invention is not limited to the use of the instrument, and a simpler instrument is used. Can also be implemented. For example, in the above specific example, the guide groove and the sample introduction groove have a two-stage structure, but the guide groove is not particularly necessary, and it is also possible to use a substrate in which only the sample introduction groove is formed on the substrate surface. . In this case, since there is no guide groove, the operator aligns the sample introduction groove with the dry gel-supported body based on visual observation. In this case as well, it is preferable to perform a hydrophilization treatment on at least the bottom surface of the sample introduction groove as described above. Furthermore, a sample provided with a hydrophilic treatment region having substantially the same size and shape as the sample introduction region on a hydrophobic plane without using a sample introduction groove should be used in the same manner as the sample introduction groove. Can do. In this case, when the sample solution is dropped on the hydrophilic treatment region, the sample solution spreads over the entire hydrophilic treatment region, but does not exit on the hydrophobic surface. For this reason, since the sample solution is held in a state where the cross-section is raised in a semicircular or semi-elliptical shape on the hydrophilization treatment region, the sample solution thus held can be brought into contact with the dried gel. The method of the invention can be carried out.

  Hereinafter, the present invention will be described more specifically based on examples and comparative examples. However, the present invention is not limited to the following examples.

  Prior to describing each example, an experimental technique common to each example will be described.

1. Preparation of mouse liver lysate
The mouse liver tissue stored at −80 ° C. was finely crushed with a scalpel blade or the like, and 0.4 g was placed in a Teflon homogenizer (Teflon is a registered trademark). 2 ml of Lysis buffer (50 mM Tris-HCl (pH 7.6), 20% Glycerol, 0.3 M NaCl, Protease inhibitor cocktail) was added and homogenized under ice cooling. Centrifugation was performed at 1000 × g at 4 ° C. for 10 minutes, and the supernatant was recovered and further centrifuged at 15,000 × g at 4 ° C. for 30 minutes. The supernatant fraction was collected and filtered through a φ0.45 μm filter.

2. Desalting and purification of protein samples The mouse liver lysate obtained in 1 above contains salts and chromosomal DNA that interfere with two-dimensional electrophoresis. In order to remove them, 2-D Clean-Up Kit (trade name, GE Healthcare Bioscience Co., Ltd.) was used. A sample (<100 μg, <100 μL) was added with 300 μL of a precipitant and stirred, and then allowed to stand on ice for 15 minutes. An additional 300 μL of co-precipitant was added and centrifuged at 14,000 × g for 5 min at 0 ° C. To the precipitate, 40 μL of a coprecipitation agent was added and allowed to stand on ice for 15 minutes, and then centrifuged at 14,000 × g for 5 minutes at 0 ° C. to collect the precipitate. After adding 25 μL of pure water, the precipitate was washed by centrifugation at 14,000 × g for 5 minutes at 0 ° C. After adding 1 mL of wash buffer and 5 μL of wash additive, the mixture was stirred and allowed to stand at −20 ° C. for 2 hours. The precipitate was collected by centrifugation at 14,000 × g for 5 minutes at 0 ° C., and then air-dried at room temperature for about 5 minutes. The obtained precipitate was dissolved in a swelling solution to obtain a sample for two-dimensional electrophoresis (for sample introduction into a dry IPG gel).

3. Protein quantification Protein quantification was performed using 2-D Quant Kit (trade name, GE Healthcare Biosciences). First, standard bovine serum albumin (BSA) was diluted as shown in Table 1 below. Samples were placed in two tubes at 1 to 50 μl each so as to be about 0.5 to 50 μg. Vortexed simply and well by adding 500 μl of coprecipitant. After centrifugation at 10,000 × g for 5 minutes, the supernatant was removed. 100 μl of copper solution was added followed by 400 μl of distilled water. Furthermore, 1 ml of a working color reagent was added and mixed instantaneously. Incubated for 15-20 minutes at room temperature and absorbance was measured at 480 nm. Samples were quantified by writing a standard curve based on the absorbance of standard BSA.

4. Sypro Ruby (trade name, Invitrogen Co., Ltd.) staining method After electrophoresis, the gel was shaken in 10% methanol / 7% acetic acid aqueous solution (about 100 mL) for 1 hour. The solution was replaced with SyproRuby staining solution (trade name, about 50 mL) and shaken for 4 hours or more. When it was desired to view with high sensitivity, it was stained overnight. After staining, after shaking in 10% methanol / 7% acetic acid aqueous solution (about 100 mL) for 30 minutes, fluorescence detection (em. 480 nm / ex. 680 nm) was performed with ProXPRESS (trade name, PerkinElmer Co., Ltd.) .

5. Reproducibility evaluation of sample introduction groove The reproducibility evaluation of the sample introduction groove was evaluated using 1.2 mm × 52 mm IPG and 48 mm × 52 mm SDS-PAGE gel. Detection and analysis were performed as in Method 5.

6. Two-dimensional electrophoresis Two-dimensional electrophoresis uses Protian IEF cell and criterion Dodeca cell (both trade names, Bio-Rad Laboratories, Inc.) and follows the standard protocols described in the instructions for use of these products. I went. The first dimension isoelectric point gel and the second dimension SDS-PAGE gel are Bio-Rad Laboratories 7cm IPG ReadyStrip pH 3-10 (trade name) and 12% acrylamide ready gel J (trade name). used. For protein staining after two-dimensional electrophoresis, SYPRO Ruby (trade name, Invitrogen) was used. Mark 12 (trade name, Invitrogen) was used as a marker.

7. Comparison of reproducibility of fluorescence intensity of protein spots on 2D electrophoresis images Detection and analysis of 2D electrophoresis spots were performed using ProFINDER (trade name, PerkinElmer Co., Ltd.) analysis software. The coefficient of variation was obtained from the fluorescence intensity of the spot obtained with ProXPRESS (trade name, Perkin Elmer Co., Ltd.), and the reproducibility of the spots was compared by comparing the coefficient of variation.

8. Sample introduction groove preparation The tool was prepared using a cutting machine. An acrylic material was used for the substrate.

9. Hydrophilic film coating The hydrophilic film (SiOx film) was formed using a plasma polymerization apparatus. The plastic substrate was placed in the polymerization chamber of the plasma polymerization apparatus, and the pressure was reduced until the ultimate vacuum in the polymerization chamber reached 1.5 × 10 ⁻³ Pascal. Next, hexamethyldisiloxane (HMDS) and oxygen were simultaneously introduced into the polymerization chamber. The flow rates of HMDS and oxygen were 5 sccm and 100 sccm, respectively, and plasma was generated at a plasma output of 300 W for 120 seconds to form a film.

Example 1
1. Manufacture of instrument A guide groove and a sample introduction groove similar to those shown in FIGS. 1 and 2 were formed in an instrument body made of an acrylic plate. The width of the guide groove opening (in the direction perpendicular to the longitudinal direction) is 1.8 mm, the bottom width is 1.4 mm, the depth is 1.0 mm, and the length is 53 mm. The corners on both sides of the direction are rounded. The sample introduction groove was formed at the center in the width direction of the guide groove, and had a width of 0.6 mm, a length of 52 mm, and a depth of 0.3 mm. The angle on both sides in the width direction of the bottom surface of the sample introduction groove was about 90 degrees.

  A plurality of the above devices were prepared, and a hydrophilic film coating was applied to a part of the bottom and side surfaces of the sample introduction groove by the method described above. The film thickness at this time was estimated to be about 70 nm, and the contact angle of the SiOx film obtained at this time was measured with a contact angle meter. As a result, it was 6.9 ° and became highly hydrophilic.

2. Introduction of sample The dry gel with plastic film used was a commercial IPG with a length of 52 mm, a width of 3 mm, and a thickness of 0.1 mm cut along the longitudinal direction to a width of 1 mm. The size of the dried gel is 52mm x 1mm x 0.1mm). A support made of a plastic block having the same length and width as the dried gel with a plastic film and a height of 14 mm was bonded to the plastic film of the dried gel with a plastic film.

  10 μL of the sample solution described above was dropped at one location near the center of the sample introduction groove. When the sample introduction groove was not hydrophilized, the sample solution formed droplets and did not spread evenly. However, when the sample introduction groove was hydrophilized, the sample solution was uniformly distributed throughout the sample introduction groove. Spread. The following experiment was performed using a sample introduction groove subjected to a hydrophilic treatment. The sample solution was filled to the upper end of the sample introduction groove with the 10 μL of the sample solution. The sample introduction and subsequent operations were performed at room temperature unless otherwise specified.

  The above-mentioned support with dry gel was inserted into the guide groove and placed on the bottom surface of the guide groove. Since the shape and size of the dry gel and the shape and size of the sample introduction groove were the same, the sample introduction groove was closed with the dry gel, whereby the dry gel was in direct contact with the sample solution. This state was left for 5 minutes. As a result, the area of the dry gel in contact with the sample solution swelled and entered the sample introduction groove. When the support with dry gel was taken out from the guide groove, no sample solution remained in the sample introduction groove, and substantially all of the sample solution was absorbed by the gel. Next, the groove (dimension 55mm x 29mm x 1.15mm) is filled with swelling liquid (composition: 8M urea, 2M thiourea, 4% CHAPS, 20mM DTT, 0.5% amphoteric electrolyte), and into this groove The support with gel was inserted and left for 5 minutes to completely swell the gel.

3. Second Dimensional Electrophoresis As described above, the gel into which 2 μg of the mouse liver lytic protein sample was introduced was equilibrated with SDS and electrophoresed on an SDS-PAGE gel for 4 minutes. The total fluorescence intensity of the obtained protein band was measured with Quantity one (trade name, Bio-Rad Laboratories Co., Ltd.).

4. Evaluation of reproducibility The above operation was repeated 4 times, and the average value, standard deviation and coefficient of variation of the total fluorescence intensity were calculated. As a result, the average of the total fluorescence intensity (x10 ¹³ ) was 9.89, the standard deviation (x10 ¹² ) was 6.71, and the coefficient of variation (%) was 6.79.

Comparative Examples 1 and 2
The IPG with plastic film (Comparative Example 1) and the IPG with plastic film (Comparative Example 2) of Company B, both commercially available from Company A, were subjected to IPG gel swelling (protein sample introduction) according to the respective standard methods. The gel was swollen using a solution obtained by dissolving the above protein sample in a swelling solution. All IPGs were 70 mm x 3 mm x 0.1 mm in size.

  In Company A's device (Comparative Example 1), add 155 μl of the specified swelling solution containing 2 μg of mouse liver soluble protein to the swelling cassette (Company A) and add 70 mm 3-10 L IPG (Company A) gel. The sample was charged and allowed to stand at room temperature for 8 hours (Comparative Example 1). This swelling cassette is a substrate with a groove having a rectangular cross section. The groove dimensions are width 5.4 mm x length 82.2 mm x depth 6.38 mm, and the groove width is gel. It is 1.8 times the width.

  Similarly, in the Company B apparatus (Comparative Example 2), 125 μl of a swelling solution containing 2 μg of mouse liver soluble protein was added to the focusing tray (Company B), and then a 70 mm 3-10 L IPG (Company B) gel was added. It was inserted and left at 20 ° C. for 16 hours (Comparative Example 2). This focusing tray also has a groove with a rectangular cross section in the substrate, the groove dimensions are 3.7 mm wide x 79.5 mm long x 1.15 mm deep, and the groove width is the same as that of the gel. It is 1.2 times the width.

  When the gel was removed from the groove after the swelling treatment, the swelling liquid remained in the groove. Subsequently, second-dimensional electrophoresis was performed in the same manner as in Example 1, and the total fluorescence intensity was measured.

  As in Example 1, the above operation was repeated four times, and the total fluorescence intensity was measured, and the average value, standard deviation, and coefficient of variation were calculated. The results are shown in Table 2 below.

  As shown in Table 2, in Comparative Examples 1 and 2 using commercially available products, the coefficient of variation of the total fluorescence intensity exceeds 20%. On the other hand, as described above, in Example 1 of the present invention, the variation coefficient is 6.79%, which indicates that the reproducibility is higher than those of Comparative Examples 1 and 2.

Example 2
In the method of Example 1, when the support with dry gel was placed on the bottom surface of the guide groove, air bubbles sometimes entered between the dry gel and the sample solution. Therefore, in order to prevent bubbles from entering, the length of the sample introduction groove was set to 54 mm, and the sample was introduced in the same manner as in Example 1 except for the other conditions. In addition, when mounting a support body with a dry gel, the support body with a dry gel was mounted in the position where the part which is not coat | covered with a dry gel each arises in the both ends of a sample introduction groove | channel. As a result, no bubbles entered between the gel and the sample solution.

Example 3
In Example 3, 10 μl of a sample solution containing 20 μg of mouse liver soluble protein was introduced into a gel in the same manner as in Example 1, and two-dimensional electrophoresis was performed. In Comparative Example 3, 125 μl of sample solution containing 20 μg of mouse liver soluble protein was subjected to two-dimensional electrophoresis by the method described above. Protein spots on the second-dimensional electrophoresis gel were detected, seven main protein spots were selected, and the fluorescence intensity of each was measured.

  The above operation was repeated four times, the fluorescence intensity was measured for each spot, and the average value, standard deviation, and coefficient of variation were calculated. The results are shown in Table 3 below.

  As is apparent from Table 3, it can be seen that in most spots, the method of Example 3 of the present invention has a smaller coefficient of variation than Comparative Example 3 using a commercially available product, and is excellent in reproducibility as a whole.

Example 4
In order to confirm that the sample can be uniformly introduced into the IPG gel in a short time, an instrument with 5 narrow grooves was prepared (Fig. 3-1), and 0.01% Coomassie was placed in the 4 narrow grooves. A sample solution mixed with Brilliant Blue R-250 dye was added. A plastic block with a 37 mm × 5 mm dry IPG gel sheet attached is brought into contact with the narrow groove, and the result after 5 minutes is shown in FIG. As a result of adding the sample solution to four of the five grooves, the sample solution was introduced into the IPG gel in the shape of the groove. The distance between the end faces of the groove was narrow 0.3 mm, and the distance of the IPG gel into which the sample was introduced was about 0.2 mm. However, since the pigment | dye was able to be confirmed slightly in the clearance gap, it seemed that the solution leaked a little. However, the leakage of the solution could be prevented by sandwiching a groove in which the sample solution was not placed. It was confirmed that the sample solution penetrated while the contact surface between the IPG gel and the sample solution was maintained.

  Furthermore, in order to confirm whether the same sample introduction is possible even if the narrow groove is rectangular, a rectangular narrow groove as shown in FIG. 3-3 was prepared and a sample introduction experiment into the IPG gel was performed. A sample solution containing 0.01% Orange G dye (trade name, Nacalai Tesque) was added to the linear groove next to the rectangular groove. As a result, the sample solution was introduced into the IPG gel with the same shape as the rectangular narrow groove (FIGS. 3-4). Similarly, the rectangular groove was able to penetrate the sample solution while maintaining the contact surface between the IPG gel and the sample solution.

It is a typical perspective view for demonstrating one preferable specific example of the method of this invention using an instrument. It is the schematic cross section which cut | disconnected the instrument shown in Example 1 in the direction orthogonal to a longitudinal direction. It is a top view of an instrument having five sample introduction grooves. FIG. 2 is a photograph of an IPG gel after introducing a sample solution with 0.01% Coomassie Brilliant Blue R-250 dye added to the third and fifth sample introduction grooves from the top of the instrument shown in FIG. 1. It is a top view of the instrument with a rectangular and straight sample introduction groove. Fig. 3-3 is a photograph of an IPG gel after introducing a sample solution containing 0.01% Coomassie Brilliant Blue R-250 dye in the rectangular groove and 0.01% Orange G dye in the straight groove. . The contact time between the sample solution and the dried IPG gel is 5 minutes.

Claims (15)

  A method for introducing a sample solution containing a sample to be electrophoresed into an elongate strip-shaped electrophoresis drying medium, wherein the sample introduction is at least a part of a sample introduction surface which is one surface of the drying medium. Including directly contacting the region with the sample solution, wherein the sample introduction region is a region extending over substantially the entire length of the sample introduction surface, and the sample solution swells the entire drying medium. Less than the amount that can be absorbed, causing the drying medium to absorb substantially all of the sample solution to partially swell the drying medium, and then swelling the entire drying medium with a swelling liquid; A method for introducing a sample into a drying medium for electrophoresis.   The method according to claim 1, wherein the drying medium is directly or indirectly supported on a rigid support on one side.   The method according to claim 1 or 2, wherein both ends of the drying medium in the longitudinal direction are not in contact with the sample solution.   The method according to any one of claims 1 to 3, wherein the support is a square bar-shaped plastic block.   The method according to claim 1, wherein the drying medium is a dry gel.   The method according to any one of claims 1 to 5, wherein one side of the drying medium is bonded to a plastic film, and the plastic film is supported on the support.   The method according to claim 1, wherein the drying medium is a first-dimensional dry gel for two-dimensional electrophoresis.   The method according to claim 7, wherein the dry gel is a pH gradient gel for isoelectric focusing. The sample solution is accommodated in a sample introduction groove having a planar shape viewed from above having substantially the same size and shape as the sample introduction region, and the sample solution accommodated in the sample introduction groove is stored in the sample introduction region. 9. The method according to any one of claims 1 to 8, wherein the method is brought into contact with.   10. The method according to claim 9, wherein at least substantially the entire bottom surface of the sample introduction groove is hydrophilized.   The sample introduction groove is provided in a bottom surface of a guide groove into which the support is inserted, and the sample solution is brought into contact with the sample introduction region in a state where the support is inserted into the guide groove. 11. The method according to any one of items 10.   An instrument for carrying out the method according to claim 9, comprising an instrument main body and the sample introduction groove provided in the instrument main body, for introducing a sample into a drying medium for electrophoresis.   The instrument according to claim 12, wherein at least substantially the entire bottom surface of the sample introduction groove is subjected to a hydrophilic treatment.   The instrument according to claim 13, wherein both ends of the sample introduction groove are not hydrophilized.   The instrument according to any one of claims 12 to 14, wherein the sample introduction groove is provided in a bottom surface of a guide groove into which a support body that supports the drying medium is inserted.

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