CN105504156A - Composite gel for gel electrophoresis and preparing method of composite gel - Google Patents
Composite gel for gel electrophoresis and preparing method of composite gel Download PDFInfo
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- CN105504156A CN105504156A CN201610005778.0A CN201610005778A CN105504156A CN 105504156 A CN105504156 A CN 105504156A CN 201610005778 A CN201610005778 A CN 201610005778A CN 105504156 A CN105504156 A CN 105504156A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/56—Acrylamide; Methacrylamide
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2333/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers
- C08J2333/24—Homopolymers or copolymers of amides or imides
- C08J2333/26—Homopolymers or copolymers of acrylamide or methacrylamide
Abstract
The invention discloses a composite gel for gel electrophoresis and a preparing method of the composite gel. The preparing method includes the following steps that acrylamide and a cross-linking agent are added into water and mixed to be even, and then a monomer, a buffer solution, a denaturing agent, an initiator and a catalyst are added to obtain a composite gel system; the composite gel system is poured into a gel frame, subjected to liquid sealing and reacted, and the composite gel is obtained, wherein the material except for the acrylamide, the cross-linking agent, the monomer, the buffer solution, the denaturing agent, the initiator and the catalyst in the system is water. As added acrylic acid or added methacrylic acid is of a double-bond structure, the added acrylic acid or the added methacrylic acid and the acrylamide can be subjected to a polymerization reaction; meanwhile, carboxyl on the acrylic acid or the methacrylic acid is reacted with amidogen of the acrylamide; as the acrylic acid or the methacrylic acid serves as a cross-linking point, the resolution ratio and the mechanical performance of the composite gel can be improved. The method is simple and wide in application range, and the selection range of the raw materials is wide.
Description
Technical field
The invention belongs to the preparation field of bioseparation dielectric material, be specifically related to a kind of gel electrophoresis plural gel and preparation method thereof.
Background technology
Gel is a kind of material with three-dimensional net structure of the formation that is cross-linked with each other by monomer and linking agent.Macromolecule hydrogel material, due to the grid structure of its uniqueness, has a wide range of applications in fields such as bioseparation, analysis, clinical diagnosises.But the performance of traditional polyacrylamide gel is comparatively single, limit its development to a certain extent.At present, be more common in mechanical properties improving hydrogel performance, and considerably less at the report of the field application such as bioseparation, analysis, clinical diagnosis about composite aquogel, less see the report adopting plural gel for gel electrophoresis.
Polyacrylamide gel electrophoresis is that the one using polyacrylamide gel as supporting dielectric commonly uses electrophoretic technique, is widely used in the fields such as bioseparation, analysis, clinical diagnosis.In electrophoresis process, protein can keep good working condition, and separates in gradient gradually according to the molecular size range of protein.In order to increase the separating power of gel to protein, usually adopt the method for the concentration increasing acrylamide and methene acrylamide.But the method for simple increase concentration often causes the mechanical property of gel to be deteriorated, (complete electrophoresis process comprises the electrophoresis of gel, fixing, dyeing and decolouring to make gel easy breakage in electrophoresis process can not complete even whole electrophoresis process, any step all can cause the breakage of gel, thus causes the failure of test.Only have the good gel of mechanical property could avoid this point to the full extent, i.e. the gel of lower concentration.But the resolving power of the gel of lower concentration is poor, the requirement of test can not be met).Meanwhile, even if increase the concentration of acrylamide and methene acrylamide, often also can only isolated molecule amount at the protein of 250 ~ 10kDa.
Summary of the invention
The problem improved is need for above-mentioned traditional cross-linked hydrogel resolving power, the invention provides a kind of gel electrophoresis plural gel and preparation method thereof, be specially: the acrylic or methacrylic acid added in acrylamide has double bond structure, can with acrylamide polymerization reaction take place, carboxyl simultaneously in acrylic or methacrylic acid and the amido of acrylamide react, because cross-linking set is served as in acrylic or methacrylic acid, resolving power and the mechanical property of plural gel can be improved.The appearance of plural gel then improves above-mentioned technical problem to a great extent.Plural gel is while improving its mechanical property, and can improve the separating power to protein preferably, especially molecular weight is greater than the protein of 250kDa.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the invention provides a kind of crosslinking structure, shown in 1:
Formula 1;
Wherein, the numerical value representated by e, f, g, h, i, j, k, l, m, n, o, p is identical or different, is and is greater than zero or null value;
R represents H or methyl.
Second aspect, the invention provides a kind of preparation method of described crosslinking structure, described preparation method is specially: under buffer conditions, and acrylamide and monomer, linking agent react and get final product;
Wherein, described monomer is acrylic or methacrylic acid; Described linking agent is N, N '-methylene-bisacrylamide.
Preferably, described damping fluid is 1.5MTris-HCl solution, pH8.8.
The third aspect, the invention provides the gel electrophoresis plural gel containing described crosslinking structure.
Fourth aspect, the invention provides a kind of preparation method of described gel electrophoresis plural gel, comprises the following steps:
Acrylamide, linking agent are added to the water, after mixing, add monomer, damping fluid, denaturing agent, initiator, catalyzer obtain composite gel system;
Poured into by described composite gel system in gel frame, fluid-tight is reacted, and obtains plural gel;
Wherein, described monomer is acrylic or methacrylic acid; Described linking agent is N, N '-methylene-bisacrylamide;
Part in described system except acrylamide, linking agent, monomer, damping fluid, denaturing agent, initiator, catalyzer is water.
Preferably, the quality of described acrylamide accounts for 6 ~ 15% of system total mass; The quality of described linking agent accounts for 0.15 ~ 0.5% of system total mass; The quality of described monomer accounts for 0.06 ~ 3% of system total mass.
Preferably, described damping fluid is 1.5MTris-HCl solution, pH8.8; The volume of described damping fluid accounts for 20 ~ 40% of system cumulative volume.
Preferably, described denaturing agent is sodium lauryl sulphate (SDS) solution of concentration 10%; The volume of described denaturing agent accounts for 0.5 ~ 1.5% of system cumulative volume.
Preferably, described initiator is ammonium persulphate (APS) solution of concentration 10%; The volume of described initiator accounts for 0.5 ~ 2% of system cumulative volume.
Preferably, described catalyzer is Tetramethyl Ethylene Diamine (TEMED); The volume of described catalyzer accounts for 0.03 ~ 0.1% of system cumulative volume.
Preferably, described water is ultrapure water.
Preferably, the mode of described mixing is for stirring.
Preferably, be incorporated as described in and add successively.
Preferably, the time of described reaction is 2 ~ 5 hours.
It is different from the mechanism of conventional gel that the present invention prepares gained gel:
The following reaction formula A of reaction formula in the preparation of conventional propylene acrylamide gel:
The following reaction formula B of reaction formula (for vinylformic acid) in acrylamide plural gel preparation of the present invention:
By more known plural gel of the present invention containing, for example lower four kinds of crosslinking structure unit (following structural units is the fractionation of above-mentioned crosslinking structure), and traditional polyacrylamide gel only has formula d crosslinking structure unit:
Wherein, the numerical value representated by a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p is identical or different, is and is greater than zero or null value;
The acrylic or methacrylic acid added in acrylamide of the present invention has double bond structure, can with acrylamide polymerization reaction take place, carboxyl simultaneously in acrylic or methacrylic acid and the amido of acrylamide react, because cross-linking set is served as in acrylic or methacrylic acid, resolving power and the mechanical property of plural gel can be improved.
Compared with prior art, the present invention possesses following beneficial effect:
1, by adding acrylic or methacrylic acid in acrylamide, resolving power and the mechanical property of plural gel can be improved;
2, plural gel and traditional gel phase ratio, mechanical property improves 5 ~ 10 times (usually at about 8 times), and resolving power improves 5 ~ 10 times;
3, the preparation method of gel electrophoresis plural gel provided by the invention, the method is simple, and material choice scope is large.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1, Fig. 2 are the contrast effect figure of plural gel of the present invention and traditional polyacrylamide gel;
Fig. 3 is plural gel containing crosslinking structure of the present invention and conventional gel infrared spectrum.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment provides a kind of gel electrophoresis plural gel and preparation method thereof, specific as follows:
By 0.6g acrylamide and 0.016gN, N '-methylene-bisacrylamide is dissolved in 5.3ml ultrapure water, 0.03g vinylformic acid, 2.5mlTris-HCl (pH8.8), 0.1mlSDS (10%), 0.1mlAPS (10%), 0.008mlTEMED is added successively after abundant stirring, to stir and after being settled to 10ml, pour in the slide mould on gum-making rack, fluid-tight, terminated reaction through 2 hours, can obtain gel electrophoresis plural gel.
embodiment 2
By 0.6g acrylamide and 0.016gN, N '-methylene-bisacrylamide is dissolved in 5.3ml ultrapure water, 0.06g vinylformic acid, 2.5mlTris-HCl (pH8.8), 0.1mlSDS (10%), 0.1mlAPS (10%), 0.008mlTEMED is added successively after abundant stirring, to stir and after being settled to 10ml, pour in the slide mould on gum-making rack, fluid-tight, terminated reaction through 3 hours, can obtain gel electrophoresis plural gel.
embodiment 3
By 0.6g acrylamide and 0.016gN, N '-methylene-bisacrylamide is dissolved in 5.3ml ultrapure water, 0.03g methacrylic acid, 2.5mlTris-HCl (pH8.8), 0.1mlSDS (10%), 0.1mlAPS (10%), 0.008mlTEMED is added successively after abundant stirring, to stir and after being settled to 10ml, pour in the slide mould on gum-making rack, fluid-tight, terminated reaction through 4 hours, can obtain gel electrophoresis plural gel.
embodiment 4
By 0.6g acrylamide and 0.016gN, N '-methylene-bisacrylamide is dissolved in 5.3ml ultrapure water, 0.06g methacrylic acid, 2.5mlTris-HCl (pH8.8), 0.1mlSDS (10%), 0.1mlAPS (10%), 0.008mlTEMED is added successively after abundant stirring, to stir and after being settled to 10ml, pour in the slide mould on gum-making rack, fluid-tight, terminated reaction through 5 hours, can obtain gel electrophoresis plural gel.
Embodiments of the invention all can actualizing technology effect in following ranges:
The quality of described acrylamide accounts for 6 ~ 15% of system total mass; The quality of described linking agent accounts for 0.15 ~ 0.5% of system total mass; The quality of described monomer accounts for 0.06 ~ 3% of system total mass; The volume of described damping fluid accounts for 20 ~ 40% of system cumulative volume; The volume of described denaturing agent accounts for 0.5 ~ 1.5% of system cumulative volume; The volume of described initiator accounts for 0.5 ~ 2% of system cumulative volume; The volume of described catalyzer accounts for 0.03 ~ 0.1% of system cumulative volume.
comparative example 1 (traditional polyacrylamide gel)
By 0.6g acrylamide and 0.016gN, N '-methylene-bisacrylamide is dissolved in 5.3ml ultrapure water, 2.5mlTris-HCl (pH8.8), 0.1mlSDS (10%), 0.1mlAPS (10%), 0.008mlTEMED is added successively after abundant stirring, to stir and after being settled to 10ml, pour in the slide mould on gum-making rack, fluid-tight, terminated reaction through 3 hours, can obtain the traditional polyacrylamide gel of gel electrophoresis.
Fig. 3 A is that whole component all adds, and wherein P (AM-AA) is plural gel, and PAM is conventional gel, in figure B, n represents and does not add linking agent, n-P (AM-AA) is plural gel, n-PAM is conventional gel, the object not adding linking agent is to verify that acrylic or methacrylic acid is (because vinylformic acid and methacrylic acid are same class material, so test for vinylformic acid) on carboxyl and acrylamide react, (the acrylic or methacrylic acid added in acrylamide has double bond structure not add linking agent, can with acrylamide polymerization reaction take place, carboxyl simultaneously in acrylic or methacrylic acid and the amido of acrylamide react, because cross-linking set is served as in acrylic or methacrylic acid).The appearance at the peak of 1288cm-1 position, the change at each peak that composition graphs marks, can prove that this cross-linking set is formed.Note: single peak value can not illustrate the formation of material, only has when a specific peak value occurs, adds the change of overall peak value, could prove the formation of material.
performance test
This experiment is according to the working specification of standard SDS-PAGE electrophoresis, and carry out performance test to embodiment 1 ~ 4 gained plural gel, detected result is as following table:
The separation performance detected result (standard model) of table 1 embodiment 1 ~ 4 gained plural gel
| Molecular weight of albumen (kDa) | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| 250 | √ | √ | √ | √ |
| 150 | √ | √ | √ | √ |
| 100 | √ | √ | √ | √ |
| 75 | √ | √ | √ | √ |
| 50 | √ | √ | √ | √ |
| 37 | √ | √ | √ | √ |
| 25 | √ | √ | √ | √ |
| 20 | × | √ | × | √ |
| 15 | × | √ | × | √ |
| 10 | × | √ | × | √ |
The separation performance detected result (actual sample: saliva) of table 2 embodiment 1 ~ 4 gained plural gel
In order to contrast intuitively, plural gel of the present invention and traditional polyacrylamide gel contrast, be below that design sketch Fig. 1: figure A (comparative example 1) and figure B (embodiment 2) is the electrophorogram of plural gel to standard model, in order to confirm that the improvement of the mechanical property of plural gel has larger help to electrophoresis, different from traditional improvement resolving power, plural gel can isolate more protein band, be generally the hypotype of albumen or different sources or isolation technique can not very protein purification at present, and traditional method can only be the raising of a general scope, the protein of 100kDa can be separated to as started, increase the concentration of acrylamide and methene acrylamide, even if the protein of 10kDa can be separated to, but as in figure B, the protein of arrows can not be separated, can only be the same with in figure A, because standard model is 10 albumen, reflecting is exactly 10 bands.
Table 3 is quantized datas to Fig. 1
| Molecular weight of albumen | The gray-scale value of conventional gel | The gray-scale value of plural gel | Plural gel/conventional gel |
| 250kDa | 7.3 | 59.1 | 8.1 |
| 150kDa | 6.7 | 57.7 | 8.6 |
| 100kDa | 6.3 | 38.5 | 6.1 |
| 75kDa | 22.6 | 91.3 | 4.0 |
| 50kDa | - | 71.2 | - |
| 37kDa | - | 39.4 | - |
| 25kDa | - | 97.1 | - |
| 20kDa | - | 38.5 | - |
| 15kDa | - | 12.0 | - |
| 10kDa | - | 33.2 | - |
Scheme A in Fig. 2 and scheme C to be respectively traditional polyacrylamide gel (comparative example 1) and plural gel (embodiment 2) electrophorogram to standard protein marker
Figure B and figure D is respectively traditional polyacrylamide gel (comparative example 1) and plural gel (embodiment 2) to actual sample: the electrophorogram of saliva
In figure D, square frame 1,2 is the protein that molecular weight is greater than 250kDa, square frame 3,4 is the protein (traditional polyacrylamide gel could not isolate) of molecular weight at 100 ~ 200kDa, square frame is above that the equal proportion of square frame is below amplified, in order to see more clearly, prevent that the monitor resolution of some readers is lower not to be seen.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. a crosslinking structure, is characterized in that, shown in 1:
formula 1;
Wherein, the numerical value representated by e, f, g, h, i, j, k, l, m, n, o, p is identical or different, is and is greater than zero or null value;
R represents H or methyl.
2. a preparation method for crosslinking structure according to claim 1, it is characterized in that, described preparation method is specially: under buffer conditions, and acrylamide and monomer, linking agent react and get final product;
Wherein, described monomer is acrylic or methacrylic acid; Described linking agent is N, N '-methylene-bisacrylamide.
3. the gel electrophoresis plural gel containing crosslinking structure described in claim 1.
4. the preparation method of a plural gel according to claim 3, it is characterized in that, described preparation method is specially: acrylamide, linking agent are added to the water, and adds monomer, damping fluid, denaturing agent, initiator, catalyzer obtain composite gel system after mixing;
Poured into by described composite gel system in gel frame, fluid-tight is reacted, and obtains plural gel;
Wherein, described monomer is acrylic or methacrylic acid; Described linking agent is N, N '-methylene-bisacrylamide;
Part in described system except acrylamide, linking agent, monomer, damping fluid, denaturing agent, initiator, catalyzer is water.
5. the preparation method of gel electrophoresis plural gel according to claim 4, is characterized in that, the quality of described acrylamide accounts for 6 ~ 15% of system total mass; The quality of described linking agent accounts for 0.15 ~ 0.5% of system total mass; The quality of described monomer accounts for 0.06 ~ 3% of system total mass.
6. the preparation method of gel electrophoresis plural gel according to claim 4, is characterized in that, described damping fluid is 1.5MTris-HCl solution, pH8.8; The volume of described damping fluid accounts for 20 ~ 40% of system cumulative volume.
7. the preparation method of gel electrophoresis plural gel according to claim 4, is characterized in that, described denaturing agent is the sodium dodecyl sulfate solution of concentration 10%; The volume of described denaturing agent accounts for 0.5 ~ 1.5% of system cumulative volume.
8. the preparation method of gel electrophoresis plural gel according to claim 4, is characterized in that, described initiator is the ammonium persulfate solution of concentration 10%; The volume of described initiator accounts for 0.5 ~ 2% of system cumulative volume.
9. the preparation method of gel electrophoresis plural gel according to claim 4, is characterized in that, described catalyzer is Tetramethyl Ethylene Diamine; The volume of described catalyzer accounts for 0.03 ~ 0.1% of system cumulative volume.
10. the preparation method of gel electrophoresis plural gel according to claim 4, is characterized in that, the time of described reaction is 2 ~ 5 hours; Described being incorporated as adds successively.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107011483A (en) * | 2017-04-07 | 2017-08-04 | 上海交通大学 | A kind of cross-linked structure plural gel for being used to be separated by electrophoresis and preparation method thereof |
| CN113804667A (en) * | 2021-09-23 | 2021-12-17 | 上海交通大学 | Novel hydrogel for expandable biological sample and application thereof |
| CN114621000A (en) * | 2022-03-29 | 2022-06-14 | 山东晶盾新材料科技有限公司 | Ceramic protective material, preparation method thereof and gel electrophoresis device |
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| CN104122317A (en) * | 2014-07-01 | 2014-10-29 | 中南大学 | Application of protease graph electrophoresis detection method to rapid identification of extracellaluar protease variety of bacteria |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107011483A (en) * | 2017-04-07 | 2017-08-04 | 上海交通大学 | A kind of cross-linked structure plural gel for being used to be separated by electrophoresis and preparation method thereof |
| CN113804667A (en) * | 2021-09-23 | 2021-12-17 | 上海交通大学 | Novel hydrogel for expandable biological sample and application thereof |
| CN114621000A (en) * | 2022-03-29 | 2022-06-14 | 山东晶盾新材料科技有限公司 | Ceramic protective material, preparation method thereof and gel electrophoresis device |
| CN114621000B (en) * | 2022-03-29 | 2024-04-02 | 山东晶盾新材料科技有限公司 | Ceramic protective material, preparation method thereof and gel electrophoresis device |
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