JP6461366B2 - Polyacrylamide gel - Google Patents

Description

  The present invention relates to a strip-like, dried polyacrylamide gel having a fixed pH gradient, which is used for first-dimension isoelectric focusing in two-dimensional electrophoresis.

  Proteins in the body play a central role in life activities, and the types and amounts of proteins expressed by translation modification are sensitive to changes in the body due to various stresses such as growth processes and diseases To change. For example, by capturing changes in protein, it is possible to determine the presence and extent of a disease, which can lead to drug discovery and treatment at the individual level. For this reason, technologies for comprehensive analysis of proteins have been extensively studied. In particular, after separation based on the charge state of proteins (including peptides) by isoelectric focusing, the molecular weight of the protein is determined by slab gel electrophoresis. The two-dimensional electrophoresis method based on separation is extremely effective because it can analyze thousands of proteins simultaneously and has high resolution.

  For the first-dimension isoelectric focusing in such two-dimensional electrophoresis, agarose gel and polyacrylamide gel are generally used. Particularly, substituted or unsubstituted acrylamide (hereinafter referred to as “acrylamide monomer”). And a crosslinking agent copolymerizable with the acrylamide monomer and an acrylamide derivative copolymerizable with the acrylamide monomer and having a weakly acidic or weakly basic buffer group. The resulting polyacrylamide gel with a fixed pH gradient (hereinafter referred to as “IPG gel”) has the advantage that it exhibits a stable pH gradient during electrophoresis and a narrow pH range can be used. Yes. The strip-shaped dried IPG gel can be stored at a low temperature, can be used simply by swelling, and is easy to handle, and thus is suitable for use.

  Such a strip-shaped dried IPG gel generally forms a slab gel having a desired pH range and pH gradient on a rectangular hydrophilic plastic film, and the resulting gel is dried and applied to the surface of the dried gel. After the protective film is adhered, it is manufactured by cutting into strips of the desired width along the direction of the pH gradient.

To date, strip-like dried IPG gels having various pH ranges and lengths (lengths in the longitudinal direction) are commercially available from GE Healthcare, Bio-Rad, Sigma-Aldrich and the like. These gels are usually obtained from a polymerization solution containing acrylamide as an acrylamide monomer and N, N′-methylenebisacrylamide (BIS) as a crosslinking agent in amounts of 4.0% T and 3.0% C. The gel has a width (length in the short direction) of 3 mm or 3.3 mm. In Examples of Patent Document 1 (Japanese Patent Application Laid-Open No. 2014-92449), 5% of acrylamide and BIS as a crosslinking agent, piperazine diacrylamide (PDA) or N, N′-diallyltartal diamide (DATD) are used. A dry IPG gel in the form of a strip, cut into several mm sizes, obtained from a polymerization solution containing T or less and 10% C or less is described. An example of Patent Document 2 (Japanese Patent Laid-Open No. 2014-92450) is obtained from a polymerization solution containing acrylamide and pentamethylenebisacrylamide or BIS as a crosslinking agent in an amount of 4% T and 2.6% C. A stripped dry IPG gel is described. Furthermore, in Example 1 of Patent Document 3 (Japanese Patent Application Laid-Open No. 2006-242802), a dried IPG gel having a thickness of 0.02 mm, a width of 0.8 mm, and a length of 52 mm manufactured by Invitrogen Corporation is used. In Example 4 (Japanese Patent Laid-Open No. 2012-242084), a dried IPG cut to a size of 0.35 mm thick × 1.15 mm wide × 52 mm long, which becomes 0.5 mm thick when swollen. Although gels are used, these documents do not describe the types and amounts of acrylamide monomers and crosslinking agents used for the production of the gels. Note that the terms% T and% C in the polymerization solution represent the following meanings.

JP 2014-92449 A JP 2014-92450 A JP 2006-242802 A JP 2012-242084 A

http: // www. sharp. co. jp / sms / release / bm-100 / na_auto2d_005c. pdf

  As described above, the IPG gel has the advantage of being excellent in the resolution of isoelectric focusing. However, when the second-dimension electrophoresis is performed after isoelectric focusing using a conventional IPG gel, the protein spot in the two-dimensional electrophoresis image is perpendicular to the second-dimension electrophoresis direction. The phenomenon of stretching has been pointed out. This phenomenon is caused by a lack of concentration of proteins with respect to the isoelectric point. This lack of concentration also leads to streaking. For example, in Non-Patent Document 1 (http://www.sharp.co.jp/sms/release/bm-100/na_auto2d — 005c.pdf), acrylamide and BIS are 4.0% T, 3.0% C A two-dimensional electrophoresis image obtained by using an IPG gel obtained from a polymerization solution containing the amount of 3.6% T or the amount of 3.6% T, 2.7% C is shown. Stretching is observed in the direction perpendicular to the surface.

  In a two-dimensional electrophoresis image, generally several hundred to several thousand spots are recognized. Further, in two-dimensional electrophoresis, it is required to analyze changes in proteins that are expressed in a slight amount or whose expression level is changed according to changes in a living body caused by stress. However, when one spot extends in the direction perpendicular to the migration direction in two-dimensional electrophoresis, adjacent spots caused by different proteins may overlap, making accurate spot volume quantification impossible. . In addition, when a spot caused by a trace amount of protein that is thinly recognized in an image extends in a direction perpendicular to the migration direction, the spot becomes further thinner, and spot discovery itself becomes difficult. Moreover, it is preferable that the extension of the spot in the migration direction in two-dimensional electrophoresis is also suppressed.

  Therefore, an object of the present invention is to provide a strip-like drying used for first-dimension isoelectric focusing, which can provide stable spot separation performance by suppressing spot extension in two-dimensional electrophoresis. An IPG gel is provided.

  The inventor changed the composition of the polymerization solution to produce various types of dried IPG gels in the form of strips, and then performed isoelectric focusing using this IPG gel and then performed the second slab gel electrophoresis. The relationship between the direction of migration of the spot in the second dimension and the extension in the perpendicular direction was investigated. As a result, a group of IPG gels were found that can suppress the extension of the spots in the direction perpendicular to the migration direction of the spot as compared with the case where a commercially available IPG gel is used. As a result of further studies, it was found that the use of an IPG gel with a relatively low protein mobility suppresses the extension of the spot in the direction perpendicular to the migration direction. Unfortunately, however, it was found that using an IPG gel with a relatively low protein mobility, the spot tends to extend in the second dimensional migration direction.

  Therefore, the inventor has repeatedly studied to solve the extension of the spot in the migration direction in the second dimension. First, when a sample containing a colored isoelectric point marker protein is subjected to isoelectric focusing using a general 3 mm-wide gel, as shown in FIG. 2 (B), along the longitudinal direction of the gel. Two lines appeared, and it was found that isoelectric point marker proteins were concentrated and migrated along these lines. Since proteins other than the isoelectric point marker protein are also proteins, it is considered that they are also concentrated on two lines along the longitudinal direction of the gel. Hereinafter, a line in which proteins along the longitudinal direction of the gel are concentrated and migrated is referred to as an “electrophoresis line”. The reason why the electrophoresis line is generated is not clear at present. However, in the process of manufacturing the strip by cutting the slab gel as described above, stress along the cut surface is generated in the strip, and it is several thousand volts in isoelectric focusing. It is thought that this is because dielectric breakdown occurs in the stress portion due to the application of voltage, and current easily flows in the dielectric breakdown portion.

  Next, as the width of the IPG gel is decreased, the two migration lines gradually approach as the width of the IPG gel decreases. As shown in FIG. It turned out to be a book running line. When the IPG gel having a relatively small protein mobility is reduced to 1.5 mm or less and isoelectric focusing is performed using this IPG gel, the second slab gel electrophoresis is performed. In addition to maintaining the phenomenon that the extension of the spot in the direction perpendicular to the migration direction is suppressed as compared with the conventional IPG gel, the extension of the spot in the migration direction is also suppressed, and thus stable in two-dimensional electrophoresis. It was found that spot separation performance was obtained. From this, it is considered that the cause of the extension of the spot in the migration direction in the two-dimensional electrophoresis is the above-described two migration lines.

  Therefore, the present invention is a strip-shaped dry IPG gel used for first-dimension isoelectric focusing in two-dimensional electrophoresis, having a width of 1.5 mm or less, and this IPG gel It relates to an IPG gel characterized in that the relative mobility of a marker protein having a molecular mass of 25 kDa measured by swelling the gel is 0.91 or less. The value of the width of the gel (the length in the short direction) in the present invention means the width of the gel in a dry state. The length of the gel of the present invention (length in the longitudinal direction) and the thickness of the gel are not particularly limited as long as they do not hinder the second-dimensional electrophoresis.

  In the present invention, the relative mobility of a marker protein having a molecular mass of 25 kDa is measured by the following procedure. FIG. 1 is a diagram for explaining measurement of relative mobility, and FIG. 1A is a schematic side sectional view of a measuring apparatus. First, the dried IPG gel 1 is swollen using a swelling liquid, for example, 250 mM trishydroxymethylaminomethane (Tris) -HCl pH 8.8 swelling liquid. For swelling, a known swelling solution can be used without particular limitation, but the swelling time needs to be carried out until sufficient swelling of the IPG gel 1 is ensured. Specifically, when the dried IPG gel 1 is swollen, the IPG gel 1 absorbs the swelling liquid, and the weight of the swollen IPG gel 1 increases as the swelling time elapses. The relationship with the weight of the gel 1 is measured in advance, and the swelling is performed until the weight of the IPG gel 1 after swelling becomes substantially constant. Next, after the swollen IPG gel 1 is fixed to the bottom of the measurement container 10, a notch with a width of 0.5 mm is formed at a position about 1 cm away from the plus extreme part of the gel, and a molecule is formed in the notch. An aqueous solution containing at least a marker protein having a mass of 25 kDa, a trace amount of bromophenol blue (BPB) and agarose for fixation is introduced and solidified to form the sample portion 20. A marker protein with a molecular mass of 25 kDa can be used in a commercial product in the amount specified in the product specification. The sample part may contain a protein other than the marker protein having a molecular mass of 25 kDa.

  Next, an electrophoresis buffer containing Tris, glycine and SDS for sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE), for example, 25 mM Tris, 192 mM glycine, 0, is attached to both ends of the swollen IPG gel 1. A filter paper 30 soaked with a buffer containing 1% SDS is placed, and an electrode cylinder 20 having a semipermeable membrane 21 at the bottom is placed on the filter paper 30. The above-described SDS-PAGE electrophoresis buffer 22 is introduced into the electrode cylinder 20, and the electrode 23 connected to the power source 40 is introduced into the electrophoresis buffer 22.

Subsequently, it introduce | transduces until the IPG gel 1 which swelled mineral oil (not shown) is covered in the measurement container 10, and applies the fixed voltage of the range of 80-120V between the electrodes 23 and 23, and implements electrophoresis. By this electrophoresis, BPB and the marker protein move in the direction of arrow E. When BPB approaches the negative pole, the electrophoresis is stopped. FIG. 1B shows a schematic plan view of the IPG gel 1 when electrophoresis is stopped. The electrophoresis starting position X _0, the migration tip position X ₁ of the band 2 of BPB, by using the electrophoresis tip position X ₂ of the band 3 of a marker protein of molecular mass 25 kDa, calculates the relative mobility Rf by the following formula .

  The width of the strip-shaped dried IPG gel is 1.5 mm or less, and the relative mobility of the marker protein having a molecular mass of 25 kDa measured by swelling the IPG gel is 0.91 or less, preferably 0.71. When the first-dimension isoelectric focusing is performed using the following IPG gel of the present invention, and then the second-dimensional slab gel electrophoresis is performed, only the extension of the spots in the two-dimensional electrophoresis in the migration direction is performed. In addition, extension in the direction perpendicular to the migration direction is also suppressed, and stable spot separation performance can be obtained. The slab gel for the second dimensional electrophoresis may have a concentrated gel part and a separation gel part, but the IPG gel of the present invention suitably suppresses the extension of spots in the migration direction. The IPG gel of the present invention can be suitably used even when combined with a slab gel having only a separation gel portion.

  There is no problem with the width of the IPG gel of the present invention if it is 1.5 mm or less, but it is generally 0.5 mm or more in consideration of the ease of cutting during manufacture and the ease of handling during use.

  As the acrylamide monomer, crosslinking agent, and acrylamide derivative for producing the IPG gel of the present invention, the acrylamide monomer, crosslinking agent, and acrylamide derivative conventionally proposed for producing the IPG gel are not particularly limited. Although it can be used, it is preferred to use DATD as the cross-linking agent. By using DATD, the pore size of the IPG gel can be increased compared to the IPG gel obtained from the polymerization solution containing 4.0% T and 3.0% C of conventional acrylamide and BIS. Therefore, a high molecular weight protein can be introduced into the IPG gel of the present invention. When using DATD as the crosslinking agent, it is preferable to use a polymerization solution of 5.5% T or more, and a polymerization solution of 20.0% C or more, more preferably 30.0% C or more. From such a polymerization solution, a high-strength IPG gel can be produced with high productivity.

  When the first-dimensional isoelectric focusing is performed using the dried IPG gel of the present invention, and then the second-dimensional slab gel electrophoresis is performed, the extension of the spots in the two-dimensional electrophoresis in the migration direction is performed. And the extension in the direction perpendicular to the migration direction are suppressed, and stable spot separation performance can be obtained.

It is explanatory drawing about the measurement of a relative mobility, (A) has shown schematic side sectional drawing of the measuring apparatus, (B) has shown schematic plan view of IPG gel, respectively. It is an image showing the relationship between the width of the gel and the appearance of the migration line for IPG gels (polymerization solution; monomer is acrylamide, crosslinker is DATD, 5.5% T, 20.0% C) with different widths (A) is an image about 1.5 mm width gel, (B) is an image about 3.0 mm width gel. 2A is a two-dimensional electrophoresis image obtained using IPG gels (polymerization solution; monomer is acrylamide, crosslinker is DATD, 5.5% T, 20.0% C). It is an image obtained using an IPG gel having a width of 5 mm, and (B) is an image obtained using an IPG gel having a width of 3.0 mm. It is a two-dimensional electrophoresis image obtained using the IPG gel of the present invention (polymerization solution; monomer is acrylamide, crosslinking agent is DATD, 6.0% T, 30.0% C: gel width; 1.5 mm). . It is a two-dimensional electrophoresis image obtained using a commercially available IPG gel (polymerization solution; monomer is acrylamide, crosslinking agent is BIS, 4.0% T, 3.0% C: gel width; 3.0 mm).

  The strip-shaped dry IPG gel of the present invention used for first-dimension isoelectric focusing in two-dimensional electrophoresis has a width of 1.5 mm or less, and the molecules measured by swelling the IPG gel. The relative mobility of the marker protein having a mass of 25 kDa is 0.91 or less, preferably 0.71 or less.

  The strip-shaped dried IPG gel of the present invention comprises an acrylamide monomer, a crosslinking agent copolymerizable with the acrylamide monomer, and a weakly acidic or weakly basic buffer group copolymerizable with the acrylamide monomer. And an acrylamide derivative having a polymerization solution.

  As the acrylamide monomer, compounds conventionally known as monomers for IPG gel can be used without particular limitation. Examples thereof include acrylamide, 2-methylacrylamide, N-methylacrylamide, and N-ethylacrylamide. N, N-dimethylacrylamide, N-isopropylacrylamide, diacetone acrylamide, and N-acryloylaminopropanol. As the monomer, a single compound may be used, or two or more compounds may be mixed and used.

  As the cross-linking agent, a compound conventionally known as a cross-linking agent for IPG gel can be used without particular limitation. Examples thereof include BIS, PDA, DATD, N, N′-bisacryloylcystamine, Examples include dihydroxyethylene bisacrylamide and pentamethylene bisacrylamide. As the crosslinking agent, a single compound may be used, or two or more compounds may be mixed and used. It is preferred to use DATD as the cross-linking agent. By using DATD, the pore size of the IPG gel can be increased compared to the IPG gel obtained from the polymerization solution containing 4.0% T and 3.0% C of conventional acrylamide and BIS. Therefore, a high molecular weight protein can be introduced into the IPG gel of the present invention.

  As the acrylamide derivative, a compound conventionally known as an acrylamide derivative for an IPG gel can be used without particular limitation, and examples thereof include an acrylamide derivative having a carboxy group and an acrylamide derivative having an amino group. . These acrylamide derivatives are also known under the name Immobiline. For the polymerization solution for the IPG gel, a plurality of acrylamide derivatives having different acid dissociation constants are mixed and used depending on a desired pH range for the IPG gel. Combinations and amounts used of acrylamide derivatives are described in, for example, G. Righetti, Immobilized pH gradients: can be determined by reference to the theory and methodology (1990).

  The production of the IPG gel is generally performed by the following steps. First, an acidic polymerization solution and a basic polymerization solution containing the acrylamide monomer, the crosslinking agent, an acrylamide derivative selected depending on the desired pH range, and a solvent are prepared. The solvent of one of the acidic polymerization solution and the basic polymerization solution is water, but the solvent of the other polymerization solution is water and a solvent used for the purpose of increasing the specific gravity of the polymerization solution, preferably Is a mixed solvent with glycerol. % T and% C of the acidic side polymerization solution and the basic side polymerization solution are adjusted to have the same value. The values of% T and% C indicate that the relative mobility of the marker protein having a molecular mass of 25 kDa measured by swelling the obtained IPG gel is 0.91 or less depending on the type of acrylamide monomer and crosslinking agent used. These values can be easily determined by a preliminary experiment for preparing an IPG gel. When using DATD as the crosslinking agent, it is preferable to use a polymerization solution of 5.5% T or more, and a polymerization solution of 20.0% C or more, more preferably 30.0% C or more. From such a polymerization liquid, an IPG gel suitable for use exhibiting high strength can be produced with high productivity.

  For the purpose of improving the polymerization efficiency as necessary, the pH of the acidic side polymerization solution and the basic side polymerization solution is adjusted to neutral using an acetic acid / sodium hydroxide buffer solution, etc., and then a polymerization initiator and a polymerization accelerator are used. Add. As the polymerization initiator, ammonium persulfate can be preferably used, and as the polymerization accelerator used in combination with this, N, N, N ′, N′-tetramethylenediamine can be preferably used. .

  Next, the acidic side polymerization solution obtained using a mixer such as a gradient mixer and a static mixer and the basic side polymerization solution are mixed while continuously changing the mixing ratio, and the resulting mixture solution is a peristic pump, Using a pump such as a piezo pump or a natural fall phenomenon, the gel is filled between glass plates of a gel preparation jig having two glass plates arranged to face each other. In addition, on the surface facing the other glass plate of one glass plate in the gel preparation jig, a hydrophilic plastic film for adhering the generated gel, generally called a gel bond film, is installed in advance. The distance between the two glass plates is adjusted to, for example, about 1 mm or less so that an IPG gel with a desired thickness can be obtained. Moreover, the filling amount of the polymerization solution is adjusted within a range of, for example, 50 mm to 240 mm so that an IPG gel having a desired length can be obtained.

  After the filling of the polymerization solution is completed, the acrylamide monomer, the crosslinking agent, and the acrylamide derivative are copolymerized. In order to advance copolymerization, you may heat to 20-50 degreeC by nitrogen atmosphere as needed. After the copolymerization is completed, the slab-like gel adhered to the hydrophilic plastic is removed from the glass plate, taken out, washed with pure water, and further immersed in glycerol for washing. Then, it is dried until the water of the slab-like gel almost disappears. A plastic protective film is adhered to the gel surface after drying, and then the gel is cut along a pH gradient together with the hydrophilic plastic film and the protective film to obtain a dried IPG gel in the form of a strip. In the present invention, the cutting is performed so that the width of the strip is 1.5 mm or less. There is no problem with the width of the IPG gel of the present invention if it is 1.5 mm or less, but it is generally 0.5 mm or more in consideration of the ease of cutting during manufacture and the ease of handling during use. The resulting strip-shaped dried IPG gel can be stored at a low temperature of about -20 ° C.

  The IPG gel of the present invention is used for first-dimension isoelectric focusing in two-dimensional electrophoresis, similar to the conventional IPG gel. Conventionally known conditions can be used without particular limitation for the sample preparation conditions for isoelectric focusing, the applied voltage in isoelectric focusing, and the running conditions such as the running time. In addition, as for the equilibration conditions for the second-dimensional SDS-PAGE of the IPG gel after electrophoresis, the electrophoresis conditions such as the applied voltage and the migration time in the second-dimensional SDS-PAGE are the same as those conventionally known. It can be used without particular limitation. The slab gel for the second dimensional electrophoresis may have a concentrated gel part and a separation gel part, but the IPG gel of the present invention suitably suppresses the extension of spots in the migration direction. The IPG gel of the present invention is suitably used in combination with a slab gel having only a separation gel part. The second-dimensional electrophoresis may be performed in a state where the gel after subjecting the IPG gel of the present invention to isoelectric focusing is fixed to the end of the second-dimensional slab gel using an agarose aqueous solution, The gel after subjecting the IPG gel of the present invention to isoelectric focusing may be carried out in a state where the gel is physically pressed against the end of the second slab gel.

  The present invention will be described with reference to the following examples, but the present invention is not limited to the following examples.

(1) Production of strip-shaped dried IPG gel and confirmation of the number of migration lines Acid side polymerization containing acrylamide as an acrylamide monomer and a crosslinking agent in the amounts shown in Table 1, and further containing an acrylamide derivative and a solvent And a basic polymerization solution were prepared. The solvent for the acidic polymerization solution was water, and the solvent for the basic polymerization solution was water and glycerol. As the acrylamide derivative, a derivative made by Sigma-Aldrich was used in an amount for producing a gel having a pH of 3 to 10. A solution in which 100 mg of ammonium persulfate is dissolved in 1.0 mL of water per 7.5 mL of each polymerization solution after adjusting the acid side polymerization solution and the basic side polymerization solution to pH 7.0 using an acetic acid / sodium hydroxide buffer. 40 μL and 4 μL of N, N, N ′, N′-tetramethylenediamine were added.

  Subsequently, the acidic side polymerization solution obtained using the gradient mixer and the basic side polymerization solution were mixed while continuously changing the mixing ratio, and the obtained mixed solution was placed oppositely by a peristaltic pump. It filled between the glass plates of the gel preparation jig which has a sheet of glass plates. A hydrophilic plastic film was placed on one surface of the glass plate before filling with the polymerization liquid.

  After filling the polymerization solution, it was allowed to stand and the copolymerization proceeded. After the copolymerization was completed, the slab-like gel adhered to the hydrophilic plastic was removed from the glass plate, taken out, washed with pure water, and further immersed in glycerol for washing. Then, dry under reduced pressure overnight until the water of the slab-like gel almost disappears, and after drying, attach a plastic protective film to the gel surface and cut the gel along with the hydrophilic plastic film and protective film along the pH gradient Thus, a dried IPG gel in the form of a strip having the gel width shown in Table 2 was obtained. The IPG length was 7 cm. The resulting strip-like dried IPG gel was stored at a low temperature of about -20 ° C.

  Next, 7M urea, 2M thiourea, 4% 3- [3- (Colamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 2 mM tributylphosphine, 0.5% IPG Buffer pH3-10NL (manufactured by GE Healthcare) A solution containing a carrier ampholyte solution) mixed with an isoelectric point marker in which a peptide is fluorescently labeled is spread linearly on a swelling tray, and a strip-like dried IPG gel shown in Table 2; Commercially available strip-shaped dried IPG gel as a conventional example (manufactured by GE Healthcare, the polymerization liquid is monomer acrylamide, the crosslinking agent is BIS, 4.0% T, 3.0% C, gel width is 3. 0 mm, length is 7.0 cm), and the conventional IPG gel is 10 hours or longer. 2 hours or more is Te, to swell the IPG gel upon standing. The swollen IPG gel is set in an isoelectric focusing device, and the applied voltage is increased stepwise at 15 ° C. (250 V 30 minutes → linearly 1000 V for 30 minutes → linearly 8000 V for 90 minutes → 8000 V For 120 minutes) isoelectric focusing. About the gel after electrophoresis, the number of electrophoresis lines was evaluated with the fluorescence imager. Table 2 summarizes the results.

  FIG. 2 shows images obtained by photographing the gel after electrophoresis for the gels of Example 2 and Comparative Example 8. (A) has shown the image regarding the gel of Example 2, (B) has shown the image regarding the gel of the comparative example 8, respectively. As clearly understood from FIG. 2, the gel of Comparative Example 8 has two migration lines, whereas the gel of Example 2 has only one migration line. Moreover, from Table 2, regardless of the composition of the polymerization solution, when a strip-shaped dry IPG gel having a gel width of 1.5 mm or less is used, only one migration line is recognized, and the gel exceeds 1.5 mm. It can be seen that when a strip-shaped dried IPG gel having a width is used, two migration lines are observed.

(2) Confirmation of Relative Mobility A 250 mM Tris-HCl pH 8.8 swelling liquid is linearly spread on a swelling tray, and the strip-shaped dried IPG gel shown in Table 2 and the above-described conventional strip-shaped dried IPG gel are used. And IPG gel was sufficiently swollen, and the IPG gel after swelling was fixed to the bottom of the measurement container shown in FIG. 1 using silicone grease. In addition, the swelling was performed for each IPG gel until the time when the swelling time and the weight of the IPG gel after swelling were measured in advance and the weight of the IPG gel after swelling was substantially constant. Next, a 0.5 mm wide notch is formed at a position about 1 cm away from the plus extreme part of the gel, and a marker protein set containing a marker protein having a molecular mass of 25 kDa in this notch (Bio-Rad, Precision, Inc.) A solution in which 0.1 μL of BPB was added to a solution prepared by mixing 1: 1 (Protein Dual Color Standard) and 5% agarose aqueous solution was solidified to form a sample portion.

  Next, filter paper impregnated with SDS-PAGE electrophoresis buffer containing 25 mM Tris, 192 mM glycine, and 0.1% SDS is placed on both ends of the swollen IPG gel, and the lower part is semi-permeable on the filter paper. An electrode cylinder provided with a membrane and having the SDS-PAGE electrophoresis buffer and electrodes introduced therein was disposed. Subsequently, after introducing into the said measurement container until the IPG gel which swelled mineral oil was covered, the voltage was applied between electrodes (80V6min-> 120V), and electrophoresis was implemented. When BPB migrates to the vicinity of the negative electrode, the migration was stopped, and the relative mobility Rf was calculated from the migration start position, the migration tip position of the BPB band, and the migration protein tip position of the marker protein band with a molecular mass of 25 kDa. . Table 3 shows the relative mobility values for the IPG gel having a gel width of 1.5 mm, together with the values for the conventional IPG gel. As is apparent from Table 3, the value of relative mobility greatly changed depending on the composition of the polymerization solution used. In addition, it is thought that it is because it is hard to produce the migration line by which protein concentrates and migrates by the voltage application of 120V, but protein is migrated comparatively uniformly in the direction of a gel width, It was obtained from the same polymerization liquid The IPG gel showed approximately the same relative mobility value without depending on the gel width.

(3) Evaluation of spot of two-dimensional electrophoresis Silkworm-derived Sf9 cells were dispersed in 8M urea, 4% CHAPS, 30 mM Tis-HCl pH 8.8 swelling solution, and 14.209 μL of soluble fraction (total protein amount 50 μg), The protein was labeled with the fluorescent dye Cy5 after mixing with 1 μL of 400 μM IC5-OSu fluorescent reagent (solvent was dimethyl sulfoxide, manufactured by Dojindo Laboratories) and left on ice for 30 minutes. Next, 1 μL of a 10 mM L-lysine solution was added to this solution and allowed to stand on ice for 10 minutes to stop the labeling reaction. A protein amount of 12 μg of the obtained liquid was collected, and a swelling buffer (7M urea, 2M thiourea, 4% CHAPS, 1.2% DeStrike Reagent (manufactured by GE Healthcare), 0.5% IPG Buffer pH3- 10 NL), dilute the total liquid volume to 125 μL for an IPG gel with a gel width of 3.0 mm, and 80 μL for the other gel width IPG gel to obtain a sample liquid It was.

  The obtained sample liquid is linearly spread on a swelling tray, and in the swollen state, a strip-like dried IPG gel showing a relative mobility of a marker protein having a molecular mass of 25 kDa of 0.91 or less and the above-described conventional strip Each of the dried IPG gels was immersed and allowed to stand for 2 hours or more (maximum 16 hours) to swell the IPG gel. The swollen IPG gel is set in an isoelectric focusing device, and the applied voltage is increased stepwise at 15 ° C. (250 V 30 minutes → linearly 1000 V for 30 minutes → linearly 8000 V for 90 minutes → 8000 V For 120 minutes) isoelectric focusing.

  The IPG gel was collected from the isoelectric focusing device, introduced into a 15 mL tube, and equilibrated with buffer A (375 mM Tris-HCl pH 8.8, 6 M urea, 2% SDS, 20% glycerol, 2% dithiothreitol (DTT). )) Was introduced into the tube and shaken for 10 minutes. The equilibration buffer A was then discarded, and instead equilibration buffer B (375 mM Tris-HCl pH 8.8, 6 M urea, 2% SDS, 20% glycerol, 4.5% iodoacetamide) was introduced into the tube, and an additional 10 Shake for a minute. Then, it was washed twice with a washing solution (125 mM Tris-HCl pH 6.8, 0.1% SDS).

  Encapsulated agarose (0.5% agarose, 125 mM Tris-HCl pH 6.8, 0.1% SDS) was heated to dissolve the agarose, and then a slab gel having no concentrated gel part (monomer is acrylamide, cross-linking agent is BIS) 12.0% T) is poured into the upper end of the slab gel of the gel plate, and immediately after the equilibrated and washed IPG gel is horizontal to the position of the upper end of the slab gel in the enclosed agarose gel. Set. After the encapsulated agarose gelled, the slab gel was set in an electrophoresis tank, filled with the electrophoresis buffer (0.3% Tris, 1.44% glycine, 0.1% SDS), and voltage was applied (80V for 30 minutes) → 200V for about 2 hours), the second-dimensional electrophoresis was performed. After the electrophoresis was completed, the slab gel was set on a fluorescence image scanner together with the gel plate, and the fluorescence of Cy5 in acrylamide was scanned to obtain a two-dimensional electrophoresis image.

  FIG. 3 shows an image obtained by photographing the gel after two-dimensional electrophoresis for the experiment using the IPG gel of Example 2 and Comparative Example 8. (A) shows an image obtained using the gel of Example 2, and (B) shows an image obtained using the gel of Comparative Example 8. As can be clearly seen from FIG. 3, in the image obtained using the gel of Comparative Example 8, the spots are extended in the migration direction, whereas in the image obtained using the gel of Example 2, The extension of the spot in the migration direction is greatly suppressed. It can also be seen that there is almost no difference in the extension of the spot in the direction perpendicular to the migration direction. Table 4 summarizes the relationship between the used IPG strip gel and the extension of the spots in the migration direction. In Table 4, the case where the extension of the spot in the migration direction is about the same as that in FIG. As can be seen from Table 4, by setting the width of the IPG gel to 1.5 mm or less, the extension of spots in the migration direction in two-dimensional electrophoresis was greatly suppressed.

  4 and FIG. 5 show the concentrated gel part (monomer) after performing isoelectric focusing using the strip-shaped dried IPG gel of Example 3 or the above-described conventional strip-shaped dried IPG gel. Was subjected to second-dimensional electrophoresis using a slab gel having acrylamide, BIS was BIS, 4.0% T) and a separation gel part (monomer was acrylamide, BIS was 12.0% T). The two-dimensional electrophoresis image is shown. 4 is an image obtained using the IPG gel of Example 3, and FIG. 5 is an image obtained using the conventional IPG gel. When these figures are compared, in the image obtained using the IPG gel of Example 3, spots corresponding to the high molecular weight protein are observed to be dark, and the IPG gel of Example 3 efficiently improves the high molecular weight protein. It turns out that it absorbed well. Further, it can be seen that when the IPG gel of Example 3 was used, the extension of the spot in the direction perpendicular to the migration direction of the spot was suppressed as compared with the case where the conventional IPG gel was used. Therefore, by using the dried IPG gel in the form of a strip according to the present invention, not only the extension of the spot in the two-dimensional electrophoresis but also the extension in the direction perpendicular to the migration direction is suppressed, and the spot in the two-dimensional electrophoresis is suppressed. It was found that the separation performance of was improved.

  A strip-like dried IPG gel is provided that suppresses both extension of the spots in the two-dimensional electrophoresis in the migration direction and extension in the direction perpendicular to the migration direction.

Claims (2)

A stripped, dried polyacrylamide gel with a fixed pH gradient used for first-dimension isoelectric focusing in two-dimensional electrophoresis,
Having a width of 1.5 mm or less, and
The polyacrylamide gel is swollen , a fixed voltage in the range of 80 to 120 V is applied to the swollen gel, and a sample containing a marker protein having a molecular mass of 25 kDa and bromophenol blue is directed from the positive electrode side to the negative electrode side of the gel. The following formula was obtained from the result of the electrophoresis test
_{R f = (X 2 -X 0} ) / (X 1 -X 0)
(In the formula, Rf represents the relative mobility of the marker protein with respect to the bromophenol blue, X ₀ represents the migration start position of the marker protein and the bromophenol blue, and X ₁ represents the migration tip position of the bromophenol blue. the stands, polyacrylamide gel X ₂ is characterized in that the relative mobility Rf for the bromophenol blue of the marker protein expressed by the representative.) electrophoresis tip position of the marker protein is 0.91 or less. The polyacrylamide gel, N, N'diallyl tartar diamide Ri gel der produced using as the crosslinking agent, the relative mobility Rf is area by der of 0.39 to 0.71, according to claim 1 A polyacrylamide gel according to 1.