JP2021183956A - Composition for gel, gel, and precast gel - Google Patents

Abstract

To provide a gel for PAGE containing polyacrylamide as a main component, which is able to give an electrophoretic gel that exhibits high separability even for a high molecular weight substance and excels in storage stability, mechanical strength and handling.SOLUTION: There are provided: a composition for a gel containing a (meth) acrylamide-based monomer and a specific crosslinking agent; a gel obtained from it; and a precast gel composed of this gel.SELECTED DRAWING: Figure 4

Description

The present invention relates to a gel composition containing a (meth) acrylamide-based monomer and a specific cross-linking agent, a gel obtained from the above composition, and a precast gel comprising the above gel.

Polyacrylamide gel electrophoresis (PAGE) is an analysis / experiment that is often used to measure, identify, and purify biological substances such as enzymes and proteins, and substances that are artificially synthesized from them. This is a method (Non-Patent Document 1). PAGE uses the phenomenon that the speed at which a charged sample substance moves inside the gel depends on the molecular weight and isoelectric point of the sample substance, and the sample substance is a gap (so-called pore) in the network structure between crosslinked polyacrylamide molecular chains. ) Moves through the gel. If the molecular weight is the same, the moving speed of the sample substance varies depending on the buffering property of the gel, that is, the electrolyte and the buffering agent contained in the gel, and in SDS-PAGE of the sample substance to which SDS is added, it varies depending on the molecular weight. Further, as PAGE becomes widespread, it is required to improve the resolution, efficiency, and workability of PAGE. Therefore, materials and devices for advantageous PAGE have been proposed (Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4).

On the other hand, the problems of PAGE itself have been pointed out. Polyacrylamide gel has a particularly low resolution for substances having a particularly large molecular weight, for example, a substance having a molecular weight of 100 kDa or more. This is because the three-dimensional size of the gap between the cross-linked polyacrylamide molecular chains (the size of the mesh formed by the cross-linked polyacrylamide molecular chains, so-called pore size) existing in the gel is relatively small. It is thought that. Even if an attempt is made to increase the pore size by lowering the concentration of acrylamide, the separating ability of polyacrylamide gel for a high molecular weight sample substance does not change so much (Non-Patent Document 2). In the case of agarose gel, which is mainly used for separating nucleic acids (DNA, RNA), the pore size is too large to be suitable for separating high molecular weight proteins. When the degree of cross-linking of acrylamide is increased and the pore size is reduced, the transparency, shape stability, and water retention of the gel are impaired, and the gel does not function as an electrophoresis gel (Non-Patent Document 3).

So far, various attempts have been made to modify the three-dimensional structure of polyacrylamide itself. Among these, 2-acrylic ethylmethacrylate is an example of cross-linking polyacrylamide with another cross-linking agent instead of N, N'-methylenebisacrylamide (BIS), which is most often used as a cross-linking agent for polyacrylamide gel. Examples of using amide (Patent Document 5), an example of using an asymmetric acrylamide derivative such as 2-acrylic ethyl methacrylate (Patent Document 6), an example of using pentamethylenebisacrylamide (Patent Document 7), and the like are known. .. However, a gel material mainly composed of polyacrylamide, which exhibits high resolution even for high molecular weight substances and can provide an electrophoresis gel having excellent storage stability, mechanical strength, and handleability, is still available. Not obtained.

"Polyacrylamide Gel Electrophoresis" Maki Onda, Archives of the Society of Protein Sciences, 1, e011 (2008) "Polyacrylamide Gel Electrophoresis" A. Chrambach and D. Rodbard, Science Vol. 172, 440-451 (1971), "Electrophoresis of Small Protein in Highly Concentrated and Crosslinked Polyacrylamide Gel" William P. et al. Cambell et. al. , Analytical Biochemistry 129, 31-36 (1983)

International Publication No. 2016/002282 Japanese Unexamined Patent Publication No. 2001-159621 International Publication No. 2015/093301 Special Table 2010-502962 International Publication No. 2017/065096 US 6,197,906 B1 Japanese Unexamined Patent Publication No. 2014-92450

The present inventor has sought a gel material that enables a more functional PAGE that has not been found so far. That is, the present inventor mainly uses polyacrylamide, which exhibits high separability even for high molecular weight substances and can provide an electrophoresis gel having excellent storage stability, mechanical strength, and handleability. I tried to design a gel material.

As a result, the gel containing crosslinked poly (meth) acrylamide obtained by polymerizing a (meth) acrylamide-based monomer in the presence of a specific crosslinking agent has all of separability, mechanical strength, and handleability for high molecular weight compounds. Was found to be effective as an excellent gel for PAGE. That is, the present invention is as follows.

(Invention 1) At least one (meth) acrylamide-based monomer selected from (meth) acrylamide having or not having a substituent and a molecular weight represented by the following formula (1) of 182 or more and 1500 or less. A composition for a gel containing a cross-linking agent.

(B represents a linear or branched alkylene group having 1 or more and 4 or less carbon atoms. R1 and R2 represent a hydrogen atom (H) or a methyl group (CH ₃ ). L, m and n represent 1, 2 and 1, respectively. It is selected from 3, and l, m, and n may be different from each other, two of l, m, and n may be the same, and all of l, m, and n may be the same, and 1 ≦ l + m + n. p, q, r are 0 ≦ p, 0 ≦ q, 0 ≦ r, 0 ≦ p + q + r.)
(Invention 2) The gel composition of Invention 1 in which the cross-linking agent is at least one selected from the following compounds CL-1 and / or CL-2.

(Invention 3) A repeating unit derived from at least one (meth) acrylamide-based monomer selected from (meth) acrylamide having or not having a substituent and a molecular weight represented by the following formula (1) are present. A gel comprising crosslinked (meth) acrylamide having a unit derived from a crosslinking agent of 182 or more and 1500 or less.

(B represents a linear or branched alkylene group having 1 or more and 4 or less carbon atoms. R1 and R2 represent a hydrogen atom (H) or a methyl group (CH ₃ ). L, m and n represent 1, 2 and 1, respectively. It is selected from 3, and l, m, and n may be different from each other, two of l, m, and n may be the same, and all of l, m, and n may be the same, and 1 ≦ l + m + n. p, q, r are 0 ≦ p, 0 ≦ q, 0 ≦ r, 0 ≦ p + q + r.)
(Invention 4) The gel of Invention 3 in which the cross-linking agent is at least one selected from the following compounds CL-1 and / or CL-2.

(Invention 5) A repeating unit derived from at least one (meth) acrylamide-based monomer selected from (meth) acrylamide having or not having a substituent and a molecular weight represented by the following formula (1) are present. A precast gel comprising a cross-linked (meth) acrylamide, having a unit derived from a cross-linking agent of 182 or more and 1500 or less.

(B represents a linear or branched alkylene group having 1 or more and 4 or less carbon atoms. R1 and R2 represent a hydrogen atom (H) or a methyl group (CH ₃ ). L, m and n represent 1, 2 and 1, respectively. It is selected from 3, and l, m, and n may be different from each other, two of l, m, and n may be the same, and all of l, m, and n may be the same, and 1 ≦ l + m + n. p, q, r are 0 ≦ p, 0 ≦ q, 0 ≦ r, 0 ≦ p + q + r.)
(Invention 6) The precast gel according to claim 5, wherein the cross-linking agent is at least one selected from the following compounds CL-1 and / or CL-2.

The electrophoresis gel containing crosslinked (meth) acrylamide produced by using the gel composition of the present invention is excellent in separability, mechanical strength, and handleability for high-molecular-weight compounds.

The graph which shows the Rf value of PAGE using the gel produced in Example 5, Example 6, and Comparative Example 3. The migration pattern of PAGE using the gels produced in Example 5, Example 6, and Comparative Example 3 and their analysis results. A migration pattern of PAGE using the gels of Example 15 and Comparative Example 12. FIG. 3A shows the gel of Example 15. FIG. 3B shows the gel of Comparative Example 12. The graph which shows the Rf value of PAGE using the gel of Example 15 and Comparative Example 12. Two-dimensional PAGE migration pattern using the gels of Example 16 and Comparative Example 13. FIG. 5A shows the gel of Example 16. FIG. 5B shows the gel of Comparative Example 13. Typical spots are surrounded by a dotted line.

[Gel Composition] The gel composition of the present invention contains a (meth) acrylamide-based monomer and a specific cross-linking agent.

[(Meta) Acrylamide Monomer] In the present invention, "(meth) acrylamide" means both metaacrylamide (methacrylamide) and acrylamide. The (meth) acrylamide-based monomer means both (meth) acrylamide having a substituent and (meth) acrylamide having no substituent. Examples of such (meth) acrylamide-based monomers include (meth) acrylamide, N-methyl (meth) acrylamide, N-ethyl (meth) acrylamide, N-isopropyl (meth) acrylamide, and N, N'-dimethyl (meth). Acrylamide, N, N'-diethyl (meth) acrylamide, N- (hydroxymethyl) (meth) acrylamide, diacetone (meth) acrylamide, N'-acrylloylaminopropanol and the like can be used. As the (meth) acrylamide-based monomer used in the present invention, one or more selected from the above (meth) acrylamides can be used. The preferred compound as the (meth) acrylamide-based monomer is acrylamide. In the present invention, a (meth) acrylamide-based monomer containing acrylamide as a main component is preferably used. In this case, the above (meth) acrylamide-based monomer is 70% by weight or more, preferably 80% by weight or more, more preferably 90% by weight or more of acrylamide with respect to the total amount thereof, and (meth) other than acrylamide in the residual ratio. It is composed of acrylamides. In the present invention, a (meth) acrylamide-based monomer composed of acrylamide is more preferably used.

[Crosslinking agent] The cross-linking agent is represented by the following formula (1) and has a molecular weight of 182 or more and 1500 or less, preferably 200 or more and 1000 or less, and more preferably 200 or more and 700 or less.

The groups: B, R1, R2 and the numbers: l, m, n, p, q, r in the equations (1) and (2) are defined as follows. B represents a linear or branched alkylene group having 1 or more and 4 or less carbon atoms. R1 and R2 represent a hydrogen atom (H) or a methyl group (CH ₃ ). l, m, n may be selected from 1, 2, 3 respectively, l, m, n may be different from each other, two of l, m, n may be the same, and all of l, m, n may be the same. May be the same, and 1 ≦ l + m + n. p, q, r are 0 ≦ p, 0 ≦ q, 0 ≦ r, 0 ≦ p + q + r. Preferred p, q, r are 0 ≦ p, 0 ≦ q, 0 ≦ r, and further 1 ≦ p + q + r.
The group A is preferably represented by the following formulas (3-1), (3-2) and (3-3).

The definition of the group B in the formulas (3-1), (3-2), and (3-3) is as described above. In equations (3-1), (3-2) and (3-3), the number of repetitions s is 0 ≦ s, and in equations (3-1), (3-2) and (3-3). It is not limited as long as the molecular weight of the cross-linking agent having the group A represented is 182 or more and 1500 or less, preferably 200 or more and 1000 or less, and more preferably 200 or more and 700 or less. As the cross-linking agent used in the present invention, the following compounds: CL-1 and CL-2 are most preferable.

In the present invention, two or more of the above-mentioned cross-linking agents can be used in combination. In addition to the above-mentioned cross-linking agent, a known cross-linking agent such as BIS can be added to the gel composition of the present invention. Further, as long as the performance of the gel composition of the present invention is not impaired, additives usually used for gels can be blended without limitation. For example, thickening polysaccharides such as agarose, and water-soluble polymers such as polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, and polymethyl vinyl ether can be added to adjust the strength and elasticity of the gel.

In the composition for gel of the present invention, the ratio of the cross-linking agent to the total of the (meth) acrylamide-based monomer and the cross-linking agent is usually 1% by weight or more and 30% by weight or less, preferably 2% by weight or more and 25% by weight or less. Hereinafter, it is more preferably 3% by weight or more and 20% by weight or less.

The gel composition of the present invention can take the form of a high-concentration aqueous solution in order to facilitate gel preparation. Such an aqueous gel composition is generally referred to as a "stock solution". When the composition for gel of the present invention is produced as a stock solution, the total amount of the (meth) acrylamide-based monomer and the above-mentioned cross-linking agent is usually 10% by weight or more with respect to the total amount of the stock solution. It is dissolved in water so as to be 10% by weight or less, preferably 10% by weight or more and 50% by weight or less, and more preferably 10% by weight or more and 40% by weight or less. In this case, the stock solution, which is one form of the gel composition of the present invention, is diluted immediately before gel production. The total amount of the (meth) acrylamide-based monomer and the above-mentioned cross-linking agent in the gel composition of the present invention immediately before gel production is usually 1% by weight or more and 40% by weight or less, preferably 2% by weight or more and 30% by weight or less, and further. It is preferably in the range of 4% by weight or more and 25% by weight or less.

The gel composition of the present invention contains a polymerization initiation reagent at the time of gel production. As the polymerization initiator, a gel production reagent generally called a polymerization initiator, a polymerization accelerator, a catalyst, a radical stabilizer, or the like can be used without limitation. Typically, a polymerization initiator consisting of ammonium persulfate (APS) and N, N, N', N'-tetramethylethylenediamine (TEMED) is used. A photopolymerization initiator such as riboflavin can also be used. The concentration of the polymerization initiation reagent follows a conventional method.

Further, a known additive such as a neutral buffer can be added to the gel composition of the present invention depending on the usage mode of the gel.

[Gel containing cross-linked (meth) acrylamide] The gel of the present invention is obtained by polymerizing the (meth) acrylamide-based monomer in water in the presence of the cross-linking agent and the polymerization initiator. The gel contains cross-linked (meth) acrylamide having a repeating unit derived from the (meth) acrylamide-based monomer and a unit derived from the cross-linking agent, and the molecular chain of the cross-linked (meth) acrylamide is in water of a matrix. It is dispersed like a three-dimensional net so as to form an appropriate gap (pore).

As shown by the above formulas (1), (2), (3-1), (3-2), (3-3), compounds (CL-1) and (CL-2), the crosslinking used in the present invention. The agent has the same terminal group structure, high symmetry, and a hydrophilic chain structure: A. It is considered that such a structure uniformly causes cross-linking points in the (meth) acrylamide molecular chain in the gel of the present invention, and the molecular chains derived from the cross-linking agent are distributed without agglomeration. That is, in the gel of the present invention, it is considered that a large mesh (pore size) formed of crosslinked (meth) acrylamide molecular chains is uniformly dispersed in the gel. The pore size of the gel of the present invention is larger and more uniform than that of a conventional acrylamide gel, and exhibits excellent separation ability for high molecular weight substances. Although it is difficult to define the overall structure of the crosslinked (meth) acrylamide constituting the gel by a general formula or a standardized index, the gel of the present invention has the overall structure of the crosslinked (meth) acrylamide constituting the gel. In that respect, it differs from conventional gels.

The gel of the present invention is particularly effective for PAGE targeting high molecular weight substances. The gel of the present invention can be used without limitation in a known apparatus used for electrophoresis (PAGE).

The buffer solution, pH adjuster, specific gravity adjuster, and surfactant injected into the electrophoresis tank together with the gel of the present invention are not limited. These are appropriately selected depending on the substance to be migrated and the migration method. For example, when separating a specific molecular weight fraction from a sample consisting of a mixture of DNA and protein, a buffer solution containing a combination of an amine compound and a zwitterion compound is used. In this case, a surfactant or a chelate compound may be added. For example, PAGE buffers such as Tris-boric acid-EDTA buffer (TBE) and Tris-phosphate-EDTA buffer (TPE) can be used.

[Precast gel] The gel of the present invention can be used as a precast gel preformed according to various electrophoresis devices. The concentration and shape of the precast gel of the present invention can be variously adjusted according to the separation target, the purpose of analysis, and the type of the electrophoresis apparatus. If the precast gel of the present invention is a mass-produced type, it can be expected that the separation / analysis accuracy will be uniform, the reproducibility will be improved, and the work cost will be reduced by using various electrophoresis devices.

[Materials / Equipment] The following materials, equipment, and conditions were used. * Is a material for comparison.

(Material for gel making)
Acrylamide, cross-linking agent: CL-1 (molecular weight 328) (compounds shown in Table 1)
Crosslinking agent: CL-2 (molecular weight 282) (compounds shown in Table 1)
Crosslinker *: BIS (N, N'-methylenebisacrylamide, molecular weight 154) (compounds shown in Table 1)
-Neutral gel buffer: EzGelAce, WSE-7310 manufactured by Atto Co., Ltd.
-Polymerization initiator: 0.05% APS and 0.05% TEMED
(Drugs and devices for electrophoresis)
・ Thickness of migration plate for gel preparation: 1 mm
-Buffer for migration: 25 mM Tris-192 mM glycine-0.1% SDS
-Etrophoresis device: Rapidus mini-slab electrophoresis tank AE-6530 manufactured by Atto Co., Ltd.
-Power supply for electrophoresis: PowerStation GhibliI WSE-3100 manufactured by Atto Co., Ltd.
・ Migration conditions: 300V constant voltage, energization time 35 minutes (molecular weight marker)
・ EzProteinLadder WSE-7020 manufactured by Atto Co., Ltd.
・ EzStandard AE-1440 manufactured by Atto Co., Ltd.
^{-Thermo Fisher Scientific HiMark TM} United Protein Standard LC5688 manufactured by Thermo Fisher Scientific.
・ Chicken muscle extract ・ HeLa cell extract (staining agent) prepared by EzApply AE-1430 manufactured by Atto Co., Ltd.
・ EzStain AQua AE-1340 manufactured by Atto Co., Ltd.
(For DNA migration)
・ DNA ladder marker made by Atto EzDNA Ladder WSE-7030
・ DNA fragment Φ174 / HincII
・ DNA fragment λ / HindIII
・ Buffer solution for migration: 25 mM Tris-144 mM glycine buffer ・ Migration conditions: 20 mA constant current ・ Fluorescent reagent EzFluoroStain DNA WSE-7130 manufactured by Atto Co., Ltd.
(For isoelectric point / two-dimensional electrophoresis)
-HeLa cell extract-Etrophoresis device: Isoelectric focusing device manufactured by Atto Co., Ltd. DiscRun Ace WSE-1510
・ Ato 5-20% concentration gradient gel E-D520L
(Dyeing agent)
・ EzStain AQua AE-1340 manufactured by Atto Co., Ltd.
(Etrophoresis gel imaging device)
・ LuminoGraphIII WSE-6300, an imaging device manufactured by Atto Co., Ltd.

[Relative mobility Rf (%)] The scanner image of the gel after electrophoresis was analyzed by CS Analyzer manufactured by Ato. The relative mobility Rf (%) was calculated by the following formula.
Rf (%) = (distance from the top of the gel to each band) ÷ (distance from the top of the gel to the tip of the electrophoresis) x 100
[Production of composition for gel (stock solution)] The above materials were blended as shown in Table 2 to produce a composition for gel (stock solution) as a stock solution. The gel composition (stock solution) (Examples 1 to 4) of the present invention and the gel composition (stock solution) for comparison (Comparative Examples 1 and 2) shown in Table 2 were obtained. In Table 2, "concentration Ts" indicates the ratio of the total amount of the monomer and the cross-linking agent to the total amount of the stock solution. In Table 2, "concentration C" indicates the ratio of the cross-linking agent to the total amount of the monomer and the cross-linking agent. In Table 2, "M / CL (molar ratio)" represents the amount ratio of the monomer and the cross-linking agent contained in the stock solution (the number of moles of the monomer / the number of moles of the cross-linking agent).

[Production of composition for gel (immediately before polymerization)] The above-mentioned neutral gel buffer solution and polymerization initiator are added to the stock solutions S1, S3, S1 * shown in Table 2 to prepare a composition for gel immediately before polymerization. bottom. The gel composition immediately before polymerization shown in Table 3 (Example 5, Example 6, Comparative Example 3) was obtained. In Table 3, "concentration Tmc" indicates the ratio of the total amount of the monomer and the cross-linking agent contained in the gel composition immediately before polymerization. In Table 3, "concentration C" indicates the ratio of the cross-linking agent to the total amount of the monomer and the cross-linking agent, and the concentration C of the stock solution is maintained.

[Production of gel] The monomer of the obtained composition for gel immediately before polymerization was polymerized (gelled) in the presence of a cross-linking agent. The gels of the present invention (Examples 5 and 6) and the gels for comparison (Comparative Example 3) shown in Table 3 were obtained. In Table 3, "concentration Tp" indicates the ratio of crosslinked polyacrylamide contained in the obtained gel, and the above concentration Tmc is maintained. In Table 3, "concentration c" indicates the ratio of the cross-linking agent to the total amount of the monomer and the cross-linking agent, and the concentration C of the stock solution is maintained.

[Electrophoresis] The molecular weight marker was electrophoresed using the obtained gel, the above-mentioned device, material, and conditions. Table 3 shows the relative mobility (Rf) of the marker with respect to the molecular weight. The concentration Tp in Table 3 shows the ratio of the total amount of the monomer and the cross-linking agent to the total amount of the gel. The concentration C in Table 3 is the ratio of the cross-linking agent to the total amount of the monomer and the cross-linking agent, and is equal to the concentration C shown in the stock solution (Table 1). The molar ratio of the monomer to the cross-linking agent contained in the stock solution is maintained in the gel composition (monomer: cross-linking agent (molar) = 81: 1). FIG. 1 is a graph showing the Rf values of Example 5, Example 6, and Comparative Example 3.

[Separation ability] As shown in Table 3 and FIG. 1, the Rf values for the electrophoretic substance having a molecular weight of 100 kDa or more are larger in the values of Examples 5 and 6 than those of Comparative Example 3. In particular, in Example 5, it can be understood that the separation ability for a running substance having a molecular weight of 140 kDa or more is high.

The migration pattern of the chicken muscle extract sample lane of the same gel was analyzed with image analysis software. FIG. 2 shows a profile near 245 kDa (myosin band). FIG. 2A shows the migration pattern of the lanes used in the analysis, in which lane 1 * shows the gel of Comparative Example 3, lane 2 shows the gel of Example 5, and lane 3 shows the gel of Example 6. As shown in FIG. 2 (b), both Example 5 (using the cross-linking agent CL-1) and Example 6 (using the cross-linking agent CL-2) use Comparative Example 3 (using the cross-linking agent BIS). ) Is narrower and sharper than the peak width. That is, the migration band is clearer in Examples 5 and 6. From this, it can be seen that the high molecular weight sample can be accurately separated by the PAGE using the gel of the present invention.

[Measurement of tensile strength (M / CL: 81, Tp: 6%)] A test piece having a length of 40 mm and a width of 30 mm was cut out from the gels of Example 5, Example 6 and Comparative Example 3, and the tensile strength was measured. A dynamic viscoelasticity tester RSA-G2 manufactured by TA Instruments was used. The test was carried out at a moving speed of 1 mm / min. The average value of the measured values of the three samples was calculated. Table 3 shows the obtained tensile fracture stress (kPa) and tensile fracture strain (%). As shown in Table 3, the gel of the comparative example is inferior in tensile strength as compared with the example.

Further, the hardness of the gels of Example 5, Example 6 and Comparative Example 3 was measured. A rubber hardness tester manufactured by Asker was used. Table 3 shows the maximum needle pressing pressure (mN) of each gel. As shown in Table 3, the gel of the comparative example is harder than the gel of the example.

From these results, the gel of the present invention containing cross-linked polyacrylamide using CL-1 or CL-2 as a cross-linking agent has a higher tensile strength than the gel containing cross-linked polyacrylamide using BIS as a conventional cross-linking agent. On the other hand, it can be seen that it is tough and highly elastic. It can be said that such a gel of the present invention has a low risk of breakage during storage, transportation, and use, and is excellent in quality stability and handleability. Therefore, the gel of the present invention is suitable as a precast gel for electrophoresis.

[Measurement of tensile strength (M / CL: 81, Tp: 5% to 10%)] The cross-linking agent concentration (M / CL) was maintained at 81 according to the gel preparation methods of Example 5, Example 6 and Comparative Example 3. Then, gels having different crosslinked polyacrylamide concentrations (Tp) were prepared. For the obtained gel, the tensile fracture stress and the tensile fracture strain were measured according to the above method. The results are shown in Tables 4 and 5. Example 8 (Tp: 6%) in Table 4 is the same as Example 5 shown in Table 3. Comparative Example 5 (Tp: 6%) in Table 5 is the same as Comparative Example 3 shown in Table 3. As shown in Tables 4 and 5, the gels of Examples 7 to 10 have higher tensile strength than the gels of Comparative Examples 4 to 7, regardless of the concentration (Tp) of the crosslinked polyacrylamide contained in the gel. Excellent.

[Tensile strength (M / CL: 63, Tp: 5% to 10%)] Using the stock solutions S2, S4, S2 * shown in Table 2, the cross-linking agent concentration (M / CL) was changed to 63 to prepare gels having different crosslinked polyacrylamide concentrations (Tp). For the obtained gel, the tensile fracture stress and the tensile fracture strain were measured according to the above method. The results are shown in Tables 6 and 7. As shown in Tables 6 and 7, even when the cross-linking agent concentration (M / CL) is 63, the gel of Examples 11 to 14 is produced regardless of the concentration (Tp) of cross-linked polyacrylamide contained in the gel. Is superior in tensile strength as compared with Comparative Examples 8 to 11. From these results, it can be said that the gel of the present invention is effective as a precast gel.

[Mechanical Strength of Gel] As described above, the gel of the present invention containing cross-linked polyacrylamide using CL-1 or CL-2 as a cross-linking agent is a gel containing cross-linked polyacrylamide using BIS as a conventional cross-linking agent. It turns out that it is tougher and more elastic than that. It can be said that such a gel of the present invention has a low risk of breakage during use and is excellent in handleability. This result also shows that the gel of the present invention is effective as a precast gel.

[Storage stability (4 ° C.)] Table 8 shows the tensile fracture stress (kPa) and the tensile fracture strain (%) when the gels produced in Example 5 and Comparative Example 3 were stored for a long period of time at 4 ° C. As shown in Table 8, the gel of the present invention exhibits superior tensile strength as compared with the gel of the comparative example even when stored at 4 ° C. for 180 days. This result also shows that the gel of the present invention is effective as a precast gel.

[Storage stability (37 ° C.)] Table 9 shows the tensile fracture stress (kPa) and tensile fracture strain (%) when the gels prepared in Example 5 and Comparative Example 3 were stored in a state of being heated to 37 ° C. show. As shown in Table 9, the tensile strength of the gel produced in Example 5 is maintained or rather improved when it is stored at 37 ° C. for 14 days. On the other hand, the tensile strength of the gel produced in Comparative Example 3 tends to decrease. This result also shows that the gel of the present invention is effective as a precast gel.

[Evaluation of storage stability] It is said that the heat history received by a gel stored at 37 ° C for one day corresponds to the case where it is stored at 4 ° C for about one month. Then, it can be said that the gel of the present invention can be stored at a low temperature of about 4 ° C. for one year or more without impairing the mechanical strength. Such a gel of the present invention can be stored for a long time as a precast gel before use.

[DNA Separability] Using stock solutions S1 and S1 *, gels (Example 15, Comparative Example 12) having a concentration Tp of 7.5% and an M / CL (molar ratio) of 81 were prepared according to the above procedure. bottom. PAGE was performed using the obtained gel, the above-mentioned material for DNA migration, and an apparatus. FIG. 3 shows the gel after migration. FIG. 3A shows the gel of Example 15. FIG. 3B shows the gel of Comparative Example 12. FIG. 4 shows Rf calculated from the gel after migration. From FIGS. 3 and 4, it can be seen that the Rf of the DNA marker having a base pair number (bp) of around 500 is larger in Example 15. From this, it can be said that the gel of the present invention is also effective for DNA PAGE and has excellent separation ability for larger DNA fragments.

[Two-dimensional electrophoresis] Using stock solutions S2 and S2 *, gels having a concentration Tp of 4% and an M / CL (molar ratio) of 63 (Example 16 and Comparative Example 13) were produced. Using the obtained gel, the above-mentioned material for isoelectric point electrophoresis, and the apparatus, two-dimensional electrophoresis of O'Farrell was performed according to a conventional method. The results are shown in FIG. FIG. 5A shows the results of Example 16, and FIG. 5B shows the results of Comparative Example 13. The arrow indicates the migration direction. From FIG. 5, it can be seen that more spots corresponding to the polymer region appear in the gel of Example 16. The gel spots of Example 16 show a more focused shape. From this, it is expected that the gel of the present invention will further improve the separation accuracy of two-dimensional electrophoresis.

The gel produced using the gel composition of the present invention is useful for PAGE and shows a clear migration pattern. The gel produced by using the gel composition of the present invention is particularly excellent in the ability to separate high molecular weight proteins and DNA. Further, the gel produced by using the gel composition of the present invention is tough and has excellent elasticity regardless of the concentration, and is less likely to be damaged during work. Moreover, the gel produced by using the gel composition of the present invention is also excellent in long-term storage stability. Therefore, the gel of the present invention is useful as a precast gel. The gel produced by using the gel composition of the present invention has significantly improved performance as compared with the conventional crosslinked polyacrylamide gel for PAGE. By using the gel composition, gel, and precast gel of the present invention, it is possible to expand the range of use of PAGE, improve the analysis accuracy using PAGE, improve the workability of PAGE, and improve the work efficiency and cost of PAGE. Will be.

1 Lane of Comparative Example 3 Lane of Example 5 Lane of Example 6 Lane of Example 6 Example of spot of Example 16 Example of spot of Comparative Example 13

Claims (6)

Includes at least one (meth) acrylamide-based monomer selected from (meth) acrylamides having or not having a substituent and a cross-linking agent having a molecular weight of 182 or more and 1500 or less represented by the following formula (1). , Composition for gel.

(B represents a linear or branched alkylene group having 1 or more and 4 or less carbon atoms. R1 and R2 represent a hydrogen atom (H) or a methyl group (CH ₃ ). L, m and n represent 1, 2 and 1, respectively. It is selected from 3, and l, m, and n may be different from each other, two of l, m, and n may be the same, and all of l, m, and n may be the same, and 1 ≦ l + m + n. p, q, r are 0 ≦ p, 0 ≦ q, 0 ≦ r, 0 ≦ p + q + r.) The gel composition according to claim 1, wherein the cross-linking agent is at least one selected from the following compounds CL-1 and / or CL-2.

The repeating unit derived from at least one (meth) acrylamide-based monomer selected from (meth) acrylamide having or not having a substituent and the molecular weight represented by the following formula (1) are 182 or more and 1500 or less. A gel comprising cross-linked (meth) acrylamide, with units derived from the cross-linking agent of.

(B represents a linear or branched alkylene group having 1 or more and 4 or less carbon atoms. R1 and R2 represent a hydrogen atom (H) or a methyl group (CH ₃ ). L, m and n represent 1, 2 and 1, respectively. It is selected from 3, and l, m, and n may be different from each other, two of l, m, and n may be the same, and all of l, m, and n may be the same, and 1 ≦ l + m + n. p, q, r are 0 ≦ p, 0 ≦ q, 0 ≦ r, 0 ≦ p + q + r.) The gel according to claim 3, wherein the cross-linking agent is at least one selected from the following compounds CL-1 and / or CL-2.

The repeating unit derived from at least one (meth) acrylamide-based monomer selected from (meth) acrylamide having or not having a substituent and the molecular weight represented by the following formula (1) are 182 or more and 1500 or less. A precast gel comprising a cross-linked (meth) acrylamide, having a unit derived from the cross-linking agent of.

(B represents a linear or branched alkylene group having 1 or more and 4 or less carbon atoms. R1 and R2 represent a hydrogen atom (H) or a methyl group (CH ₃ ). L, m and n represent 1, 2 and 1, respectively. It is selected from 3, and l, m, and n may be different from each other, two of l, m, and n may be the same, and all of l, m, and n may be the same, and 1 ≦ l + m + n. p, q, r are 0 ≦ p, 0 ≦ q, 0 ≦ r, 0 ≦ p + q + r.) The precast gel according to claim 5, wherein the cross-linking agent is at least one selected from the following compounds CL-1 and / or CL-2.

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