CN102229688B - Ionic liquid- polyacrylamide gel, preparation method and purpose thereof - Google Patents

Abstract

The invention relates to an ionic liquid-polyacrylamide gel, a preparation method and a purpose thereof, and belongs to the technical field of protein separation and analysis. The ionic liquid-polyacrylamide gel comprises a monomer, a cross-linking agent, a gel buffer system, a catalyst, an accelerator and an ionic liquid, wherein the ionic liquid has cations having no more than 4 carbon atoms on an alkyl carbon chain, and has a mass concentration of 0.05-1.0%(w/v). The ionic liquid, the monomer, the cross-linking agent and the gel buffer system are mixed together, and added with the catalyst and the accelerator to be mixed uniformly; and the mixture is solidified to obtain the ionic liquid-polyacrylamide gel. The ionic liquid-polyacrylamide gel can be applied to protein electrophoretic separation, namely, an ionic liquid-sodium dodecyl sulfate-polyacrylamide gel is used for electrophoresis separation of an SDS treated protein. Especially, for different proteins having a same or a similar molecular weight, a rapid, simple, green and efficient separation thereof can be realized.

Description

A kind of ionic liquid-polyacrylamide gel, its preparation method and application

technical field

The present invention relates to a kind of ionic liquid-polyacrylamide gel (ILs-PAG), its preparation method and application, specifically, the ionic liquid (ILs) in the described ionic liquid-polyacrylamide gel is cationic upper alkyl The invention relates to an ionic liquid with a base carbon chain carbon atom number less than or equal to 4, belonging to the technical field of protein separation and analysis. the

Background technique

Since the completion of the sequencing of the human genome, proteomics research has received extensive attention. In recent years, proteomics research has been widely used in basic life sciences, medical analysis, and disease marker research. Complete proteomics analysis includes four parts: sample preparation, protein separation, protein analysis identification and characterization, among which protein separation and analysis technology is the key to proteomics research, which restricts the development of proteomics research. the

Since shapizo proposed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 1967, SDS-PAGE, as a classic protein separation and analysis technique, has been widely used in the fields of biological separation and proteomics. The basic principle of SDS-PAGE is that protein combines with sodium dodecyl sulfate (SDS) to form a negatively charged protein-SDS complex, which can eliminate the charge difference between different protein molecules, so that protein molecules can be separated in polyacrylamide condensation. The electrophoretic mobility in the gel (PAG) is no longer affected by the original charge of the protein, but mainly depends on the molecular weight of the protein, and its mobility is linearly related to the logarithm of the molecular weight of the protein. This property of SDS-PAGE can realize the effective separation of proteins, and can use the relationship between migration distance and protein molecular weight to conduct qualitative research on proteins. However, for different proteins with equal or similar molecular weights, SDS-PAGE cannot effectively separate and identify them. the

Ionic liquids, also known as room temperature ionic liquids or room temperature molten salts, refer to molten salts that are liquid at room temperature or near room temperature, and are usually composed of organic cations and inorganic anions. Ionic liquids not only have the advantages of traditional organic solvents, but also have many unique advantages, such as low vapor pressure at room temperature, non-flammability, good stability, strong solubility and high conductivity. Most importantly, ionic liquids can be designed as solvents by modifying the physicochemical properties of ionic liquids in a targeted manner by changing the species of cations and anions. Ionic liquids with special functions can also be designed by introducing some functional groups with special functions on the cations in the ionic liquids. For this reason, ionic liquids have been widely used in the field of bioseparation analysis in recent decades. the

There have been reports on related methods that have successfully used ionic liquids to improve protein separation and analysis techniques. For example, the method introduced in the article "Simultaneous separation of basic and acidic proteins using 1-butyl-3-methylimidazolium based ion liquid as dynamic coating and background electrolyte in capillary electrophoresis" (DOI 10.1002/elps.200700746, 2008, 29, , using 1-butyl-3-methylimidazolium tetrafluoroborate as an additive to the capillary electrophoresis electrolyte, successfully baseline-separated five proteins with different properties within 14 minutes. In this method, the ionic liquid can be dynamically coated on the surface of the inner wall of the capillary to change the charge property of the inner wall, thereby reducing the adsorption of the inner wall of the capillary to the basic protein. So far, there is no report on the application of ionic liquids to improve SDS-PAGE and other gel separation techniques, but the present invention just solves this problem. the

Contents of the invention

For the defect that the SDS-PAGE method has a low degree of separation of different proteins with equal or similar molecular weights, one of the objectives of the present invention is to provide an ionic liquid-polyacrylamide gel; specifically, the ionic liquid-polyacrylamide The ionic liquid in the gel is an ionic liquid with an alkyl carbon chain carbon atom number≤4 on the cation. the

The second object of the present invention is to provide a preparation method of ionic liquid-polyacrylamide gel. the

The third object of the present invention is to provide an application of ionic liquid-polyacrylamide gel, which is to use ionic liquid-polyacrylamide gel for electrophoresis analysis of proteins, i.e. ionic liquid-sodium lauryl sulfate - Polyacrylamide gel electrophoresis (ILs-SDS-PAGE). Using the electrophoresis method to electrophoretically separate proteins treated with SDS can realize fast, simple, green and efficient separation of different proteins with equal or similar molecular weights. the

For realizing the purpose of the present invention, the technical scheme adopted is as follows. the

An ionic liquid-polyacrylamide gel, the gel is composed of an ionic liquid and a polyacrylamide gel formula, that is, an ionic liquid-polyacrylamide gel, which mainly includes a monomer, a crosslinking agent, a gel The gel buffer system, catalyst and accelerator are characterized in that: ionic liquid is also included. the

Wherein, the ionic liquid is an ionic liquid with an alkyl carbon chain carbon atom number ≤ 4 on the cation, including but not limited to an imidazole ionic liquid, a pyridine ionic liquid or a pyrrolidine ionic liquid with an alkyl carbon chain carbon atom number ≤ 4 liquid. the

The mass concentration range of the ionic liquid is 0.05-1.0% (w/v). the

The polyacrylamide gel formula is a common polyacrylamide gel formula in the field of protein separation and analysis, including but not limited to the following components: monomers, cross-linking agents, gel buffer systems, catalysts and accelerators; Among them, each component includes but not limited to: monomer acrylamide, crosslinking agent N, N'-methylenebisacrylamide, gel buffer system, catalyst ammonium persulfate (APS) and accelerator N, N, N ',N'-Tetramethylethylenediamine (TEMED). Wherein the gel buffer system is one of the polyacrylamide gel buffer systems commonly used in protein separation analysis in the art, including but not limited to Tris-HCl buffer system, borax-boric acid buffer system or disodium hydrogen phosphate and Sodium dihydrogen phosphate buffer system. The mass concentration of each component is the same as the mass concentration of each component of the polyacrylamide gel formula commonly used in protein separation and analysis in this field, and is determined according to actual needs. the

The gel system of the ionic liquid-polyacrylamide gel in the present invention is a continuous gel system or a discontinuous gel system, which is the same as the polyacrylamide gel system commonly used in protein separation and analysis in the art. The mass concentration of acrylamide and N,N'-methylenebisacrylamide in polyacrylamide gel, including but not limited to continuous gel system, is 4-17% (w/v), and the pH value is 5.8-8.8; The mass concentration of acrylamide and N, N'-methylenebisacrylamide in the separating gel and stacking gel in the discontinuous gel system is 4-17% (w/v), wherein the concentration of the separating gel is greater than the concentration of the stacking gel, and the pH The value is 5.8 to 8.8. the

The mass concentration of the ionic liquid is determined according to the main molecular weight and gel concentration of the target protein to be separated. Wherein, the protein is a protein suitable for SDS-PAGE in protein separation and analysis in the art, including but not limited to protein molecular weight markers and human serum protein biological samples with a molecular weight lower than 66.2KDa. the

For the protein molecular weight marker, it is prepared by using the imidazole-based ionic liquid, pyridine-based ionic liquid or pyrrolidine-based ionic liquid with a mass concentration range of 0.05-1.0% (w/v) provided by the invention and an alkyl carbon chain with a carbon number ≤ 4 The ionic liquid-polyacrylamide gel can be successfully separated. the

For the protein in the molecular weight range of 66.2～26.0KDa in the human serum sample, use the mass concentration scope provided by the present invention to be the imidazole ionic liquid of the alkyl carbon chain carbon number≤4 of 0.05～1.0% (w/v), pyridine Compared with the parallel control experiment SDS-PAGE, the separation of ionic liquid or pyrrolidine ionic liquid-polyacrylamide gel improved the resolution of proteins and increased the number of clear protein bands in regions with similar molecular weights. Wherein, the degree of separation and the number of bands of the protein increase as the concentration of the ionic liquid increases. It is preferable to use a pyridine-based ionic liquid-polyacrylamide gel with a mass concentration of 1.0% (w/v), at which point the separated protein can obtain the best number of separated bands and degree of separation. the

For proteins with a molecular weight ranging from 26.0 to 12.0 KDa in human serum samples, use imidazoles, pyridines and pyrrolidines with a mass concentration range of 0.3 to 1.0% (w/v) of alkyl carbon chain carbon atoms ≤ 4 Liquid-polyacrylamide gel separation, compared with its parallel control experiment SDS-PAGE, improves the resolution of proteins and increases the number of clear protein bands in regions with similar molecular weights. Among them, the degree of protein separation and the number of bands increased with the increase of ionic liquid concentration. It is preferable to use a pyridine-based ionic liquid-polyacrylamide gel with a mass concentration of 1.0% (w/v), at which point the separated protein can obtain the best number of separated bands and degree of separation. the

A kind of preparation method of ionic liquid-polyacrylamide gel, described method step is as follows:

First mix the ionic liquid, monomer, cross-linking agent and gel buffer system to obtain a mixed solution, then add catalyst and accelerator to the mixed solution and mix evenly, after solidification, an ionic liquid according to the present invention can be obtained - Polyacrylamide gel. the

Wherein, the ionic liquid is an ionic liquid with an alkyl carbon chain carbon atom number ≤ 4 on the cation, including but not limited to an imidazole ionic liquid, a pyridine ionic liquid or a pyrrolidine ionic liquid with an alkyl carbon chain carbon atom number ≤ 4 liquid. the

The mass concentration range of the ionic liquid is 0.05-1.0% (w/v). the

In addition to the ionic liquid, other preparation methods of the ionic liquid-polyacrylamide gel in the present invention are general polyacrylamide gel preparation methods in the field of protein separation and analysis. Including but not limited to mixing the appropriate proportion of ionic liquid, monomer, cross-linking agent and gel buffer system to obtain a mixed solution, then adding catalyst and accelerator to the mixed solution and mixing evenly, after solidification, the present invention can be obtained Described a kind of ionic liquid-polyacrylamide gel. Including but not limited to mixing an appropriate proportion of ionic liquid, monomer acrylamide, cross-linking agent N, N'-methylenebisacrylamide and gel buffer system to obtain a mixed solution, and then catalyst ammonium persulfate and accelerator N, N, N', N'-tetramethylethylenediamine is added to the mixed solution, mixed uniformly, and after solidification, an ionic liquid-polyacrylamide gel of the present invention can be obtained. Wherein the gel buffer system is one of the polyacrylamide gel buffer systems commonly used in protein separation analysis in the art, including but not limited to Tris-HCl buffer system, borax-boric acid buffer system or disodium hydrogen phosphate and Sodium dihydrogen phosphate buffer system. The mass concentrations of monomers, cross-linking agents, gel buffer systems, catalysts and accelerators are the same as the mass concentrations of each component of the polyacrylamide gel formula commonly used in protein separation analysis in this field, and are determined according to actual needs. the

The gel system of the ionic liquid-polyacrylamide gel in the present invention is a continuous gel system or a discontinuous gel system, which is the same as the polyacrylamide gel system commonly used in protein separation and analysis in the art. The mass concentration of acrylamide and N,N'-methylenebisacrylamide in the polyacrylamide gel including but not limited to the continuous gel system is 4-17% (w/v), and the pH value is 5.8-8.8; The mass concentration of acrylamide and N, N'-methylenebisacrylamide in the separating gel and stacking gel in the discontinuous gel system is 4-17% (w/v), wherein the concentration of the separating gel is greater than the concentration of the stacking gel, and the pH The value is 5.8 to 8.8. the

The mass concentration of the ionic liquid is determined according to the main molecular weight and gel concentration of the target protein to be separated. Wherein, the protein is a protein suitable for SDS-PAGE in protein separation and analysis in the art, including but not limited to protein molecular weight markers and human serum protein biological samples with a molecular weight lower than 66.2KDa. the

For the protein molecular weight marker, it is prepared by using the imidazole-based ionic liquid, pyridine-based ionic liquid or pyrrolidine-based ionic liquid with a mass concentration range of 0.05-1.0% (w/v) provided by the invention and an alkyl carbon chain with a carbon number ≤ 4 The ionic liquid-polyacrylamide gel can be successfully separated. the

For the protein in the molecular weight range of 66.2～26.0KDa in the human serum sample, use the mass concentration scope provided by the present invention to be the imidazole ionic liquid of the alkyl carbon chain carbon number≤4 of 0.05～1.0% (w/v), pyridine Compared with the parallel control experiment SDS-PAGE, the separation of ionic liquid or pyrrolidine ionic liquid-polyacrylamide gel improved the resolution of proteins and increased the number of clear protein bands in regions with similar molecular weights. Wherein, the degree of separation and the number of bands of the protein increase as the concentration of the ionic liquid increases. It is preferable to use a pyridine-based ionic liquid-polyacrylamide gel with a mass concentration of 1.0% (w/v), at which point the separated protein can obtain the best number of separated bands and degree of separation. the

For proteins with molecular weights ranging from 26.0 to 12.0 KDa in human serum samples, use imidazoles, pyridines and pyrrolidine ions with a mass concentration range of 0.3 to 1.0% (w/v) of alkyl carbon chain carbon atoms ≤ 4 Liquid-polyacrylamide gel separation, compared with its parallel control experiment SDS-PAGE, improves the resolution of proteins and increases the number of clear protein bands in regions with similar molecular weights. Among them, the degree of protein separation and the number of bands increased with the increase of ionic liquid concentration. It is preferable to use a pyridine-based ionic liquid-polyacrylamide gel with a mass concentration of 1.0% (w/v), at which point the separated protein can obtain the best number of separated bands and degree of separation. the

An application of ionic liquid-polyacrylamide gel, the application is to use ionic liquid-polyacrylamide gel for electrophoresis analysis of protein, that is, ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. the

The steps of the electrophoresis method are as follows:

(1) prepare a kind of ionic liquid-polyacrylamide gel;

(2) SDS pretreatment protein sample;

(3) Preparation of protein electrophoresis buffer and loading;

(4) Electrophoresis;

(5) Dyeing and decolorization. the

It is characterized in that: the gel used in the electrophoresis is the ionic liquid-polyacrylamide gel of the present invention, and the rest of the electrophoresis conditions are the same as the electrophoresis conditions applicable to SDS-PAGE in protein separation and analysis in the art. the

Specifically, the electrophoresis method includes but is not limited to:

(1) Preparation of ionic liquid-polyacrylamide gel

Assemble the gel frame made of glass plates, preferably use vaseline to seal the sides and bottom of the glass plate of the gel frame to prevent leakage; if it is a continuous gel system, first mix the ionic liquid, monomer, cross-linking agent and gel buffer system to obtain a mixed solution, then add the catalyst and accelerator to the mixed solution and mix evenly to obtain the ionic liquid-polyacrylamide gel solution, inject the gel solution into the glass plate of the gel holder, and then insert the comb into the gel in the glass plate In the gel solution until the gel is solidified, pull out the comb to form the injection hole; if it is a discontinuous gel system, it is necessary to make a separating gel and a stacking gel separately, and the method is similar to the continuous gel system.

(2) Pretreatment of protein samples

The sample buffer solution is added to the protein sample, mixed evenly, heated, and then cooled to obtain a pretreated protein sample. Among them, it is preferred to heat in a water bath at 95°C for 5 minutes and cool at room temperature; the mass concentration of protein samples is preferably 0.1-0.5 mg/mL; sample buffers include but not limited to buffer systems, SDS, glycerin, bromophenol blue, and β-mercaptoethanol and water, the mass ratio of SDS to protein sample is ≥1.45:1; in the continuous gel system, the type, concentration and pH value of the sample buffer buffer system are the same as those of the ionic liquid-polyacrylamide gel system; In the discontinuous gel system, the type, concentration and pH value of the sample buffer buffer system are the same as the gel buffer system of the concentrated gel solution of the ionic liquid-polyacrylamide gel system. the

(3) Prepare protein electrophoresis buffer and load it

Assemble the electrophoresis instrument, prepare the protein electrophoresis buffer and add it to the electrophoresis tank; pour the protein electrophoresis buffer over the short plate of the glass plate of the gel holder, and use a micro-sampler to inject the protein sample pretreated in step (2) into the channel sample. the

Among them, the loading volume of the protein sample is preferably 5-10 μL. In the continuous gel system, the type, concentration and pH value of the electrophoresis buffer buffer system are the same as those of the ionic liquid-polyacrylamide gel system; in the discontinuous gel system, the type of electrophoresis buffer buffer system , concentration and pH value are the same as the gel buffer system of the concentrated gel solution of the ionic liquid-polyacrylamide gel system. the

(4) Electrophoresis

Switch on the electrophoresis apparatus for electrophoresis. Continuous gel system, using constant voltage electrophoresis. The discontinuous gel system is first concentrated with a low voltage. When the bromophenol blue reaches the separation boundary of the separating gel and the stacking gel, a relatively high voltage is used for electrophoresis separation until the bromophenol blue reaches the bottom of the gel, and the electrophoresis ends. the

Among them, in the continuous gel system, the voltage is 80-200V; in the discontinuous gel system, the voltage of the stacking gel is generally 80-140V, and the voltage of the separating gel is 100-160V. the

(5) Dyeing and decolorization

After the electrophoresis, the gel was peeled off from the glass plate, washed with water, and then stained. After the staining was completed, the gel was decolorized until the background color was removed. the

Peel the gel off the glass plate preferably in water to prevent the gel from cracking. the

The staining is a staining technique applicable in protein separation and analysis in the art, including but not limited to Coomassie Brilliant Blue R250 staining, Coomassie Brilliant Blue G250 staining or silver staining. the

The mass concentration of the ionic liquid is determined according to the main molecular weight and gel concentration of the target protein to be separated. Wherein, the protein is a protein suitable for SDS-PAGE in protein separation and analysis in the art, including but not limited to protein molecular weight markers and human serum protein biological samples with a molecular weight lower than 66.2KDa. the

For the protein molecular weight marker, it is prepared by using the imidazole-based ionic liquid, pyridine-based ionic liquid or pyrrolidine-based ionic liquid with a mass concentration range of 0.05-1.0% (w/v) provided by the invention and an alkyl carbon chain with a carbon number ≤ 4 The ionic liquid-polyacrylamide gel was successfully separated by ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. the

For the protein in the molecular weight range of 66.2～26.0KDa in the human serum sample, use the mass concentration scope provided by the present invention to be the imidazole ionic liquid of the alkyl carbon chain carbon number≤4 of 0.05～1.0% (w/v), pyridine Ionic liquid-like ionic liquid or pyrrolidine-like ionic liquid-polyacrylamide gel was carried out by ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, compared with its parallel control experiment SDS-PAGE, the separation degree of protein was improved, and the The number of clear protein bands in regions with similar molecular weights is shown. Wherein, the degree of separation and the number of bands of the protein increase as the concentration of the ionic liquid increases. It is preferable to use a pyridine-based ionic liquid-polyacrylamide gel with a mass concentration of 1.0% (w/v), at which point the separated protein obtains the best number of separated bands and degree of resolution. the

For proteins with molecular weights ranging from 26.0 to 12.0 KDa in human serum samples, use imidazoles, pyridines and pyrrolidine ions with a mass concentration range of 0.3 to 1.0% (w/v) of alkyl carbon chain carbon atoms ≤ 4 Ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out on liquid-polyacrylamide gel. Compared with the parallel control experiment SDS-PAGE, the separation of proteins was improved, and clear protein strips in regions with similar molecular weights could be increased. Bring a number. Among them, the degree of protein separation and the number of bands increased with the increase of ionic liquid concentration. It is preferable to use a pyridine-based ionic liquid-polyacrylamide gel with a mass concentration of 1.0% (w/v), at which point the separated protein can obtain the best number of separated bands and degree of separation. the

Beneficial effect

1. Simple, fast, efficient and reproducible. The present invention adds an ionic liquid with an alkyl carbon chain carbon number of ≤ 4 on the basis of the polyacrylamide gel formula to prepare an ionic liquid-polyacrylamide gel; on the basis of the SDS-PAGE method, using Ionic liquids improve the SDS-PAGE method, and use ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate proteins, which has the advantages of simplicity, speed, efficiency and good reproducibility. SDS-PAGE is a classic protein separation and analysis technique with a mature method, simple and easy to operate, and is widely used in various fields of protein research. Ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis using ionic liquids to improve SDS-PAGE is also simple and easy to operate, and compared to SDS-PAGE, ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis Gel electrophoresis separates proteins with higher resolution;

2. It is not easy to produce air bubbles during the glue making process. Since the ionic liquid-polyacrylamide gel in the present invention does not add SDS, the bubbles formed by SDS are reduced, the gel solution is conveniently transferred to the glass plate of the glue holder, and the generation of bubbles in the gel solidification process is also reduced. , making the electrophoretic environment more stable;

3. Selective separation of proteins. SDS-PAGE cannot effectively separate different proteins with equal or similar molecular weights. Using the ionic liquid-polyacrylamide gel provided by the present invention, since the ionic liquid in the ionic liquid-polyacrylamide gel can interact with proteins, thereby affecting the migration rate of proteins, the separation of proteins with similar molecular weights is realized; The ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis provided by the invention can achieve better selective separation of different proteins with equal or similar molecular weights, especially for human serum proteins with a molecular weight lower than 66.2KDa. selective separation. the

4. Security. Ionic liquids are safe and pollution-free, known as green solvents, and will not cause harm to humans and the environment;

5. Universality. The ionic liquid-polyacrylamide gel provided by the present invention and the corresponding established ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis method can be directly applied to protein separation, and are also suitable for polyacrylamide gel electrophoresis The improvement of the method is expected to become an important technology for proteomics and protein separation analysis;

6. Prevent the gel from breaking. In the ionic liquid-sodium lauryl sulfate-polyacrylamide gel electrophoresis method step (5) provided by the invention, the ionic liquid-polyacrylamide gel is peeled off from the glass plate in water, can prevent gel rupture;

7. Prevent leakage. When assembling the gel-making frame in the ionic liquid-sodium lauryl sulfate-polyacrylamide gel electrophoresis method step (1) provided by the invention, use vaseline to seal the both sides and the bottom of the glass plate of the gel-making frame to prevent liquid leakage . the

Description of drawings

Figure 1 is a schematic diagram of a method for separating proteins by ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. the

Fig. 2 is that the mass concentration of 1-ethyl-3-methylimidazolium tetrafluoroborate prepared in Example 1 is 0.05% (w/v), 0.1% (w/v), 0.2% (w/v) , 0.3% (w/v), 0.6% (w/v) and 1.0% (w/v) 1-ethyl-3-methylimidazolium tetrafluoroborate-polyacrylamide gel ([C ₂ mim]BF ₄ -PAG) electrophoresis electrophoresis separation of protein molecular weight marker electrophoresis.

Fig. 3 is that the 1-butyl-3-methylimidazolium tetrafluoroborate mass concentration that embodiment example 1 prepares is 0.05% (w/v), 0.1% (w/v), 0.2% (w/v) , 0.3% (w/v), 0.6% (w/v) and 1.0% (w/v) 1-butyl-3-methylimidazolium tetrafluoroborate-polyacrylamide gel ([C ₄ mim]BF ₄ -PAG) electrophoresis electrophoresis separation of protein molecular weight marker electrophoresis.

Fig. 4 is the brominated N-butyl-3-picoline-polyacrylamide gel with a mass concentration of brominated N-butyl-3-picoline prepared in Example 2 of 0.05% (w/v) ([C ₄ mpd]Br-PAG), N-butyl-3-methylpyrrolidine bromide with mass concentration of 0.05% (w/v) brominated N-butyl-3-methylpyrrolidine-poly Acrylamide gel ([C ₄ mprd]Br-PAG) and 1-butyl-3-methylimidazole bromide with a mass concentration of 0.05% (w/v) - The electrophoresis of ten-fold diluted human serum separated by polyacrylamide gel ([C ₄ mim]Br-PAG) and its parallel control experiment SDS-PAGE separated by ten-fold diluted human serum.

Fig. 5 is the brominated N-butyl-3-picoline-polyacrylamide gel ([C ₄ mpd ]Br-PAG), N-butyl-3-methylpyrrolidine bromide-polyacrylamide gel with a mass concentration of 0.3% ([C ₄ mprd] Br-PAG) and brominated 1-butyl-3-methylimidazole-polyacrylamide gel ([C ₄ mim]Br-PAG ) to separate the electrophoresis of ten-fold diluted human serum and its parallel control experiment SDS-PAGE to separate the electrophoresis of ten-fold diluted human serum.

Fig. 6 is the brominated N-butyl-3-picoline-polyacrylamide gel ([C ₄ mpd ]Br-PAG), N-butyl-3-methylpyrrolidine bromide-polyacrylamide gel with a mass concentration of 1.0% ([C ₄ mprd] Br-PAG) and brominated 1-butyl-3-methylimidazole-polyacrylamide gel ([C ₄ mim]Br-PAG with a mass concentration of 1.0%) ) to separate the electrophoresis of ten-fold diluted human serum and its parallel control experiment SDS-PAGE to separate the electrophoresis of ten-fold diluted human serum.

Detailed ways

In order to fully illustrate the characteristics of the present invention and the mode of carrying out the present invention, examples are given below. the

Example 1

The mass concentration of 1-ethyl-3-methylimidazolium tetrafluoroborate prepared respectively is 0.05% (w/v), 0.1% (w/v), 0.2% (w/v), 0.3% (w/v ), 0.6% (w/v) and 1.0% (w/v) 1-ethyl-3-methylimidazolium tetrafluoroborate-polyacrylamide gel ([C ₂ mim]BF ₄ -PAG) And 1-butyl-3-methylimidazolium tetrafluoroborate mass concentration is 0.05% (w/v), 0.1% (w/v), 0.2% (w/v), 0.3% (w/v) , 0.6% (w/v) and 1.0% (w/v) 1-butyl-3-methylimidazolium tetrafluoroborate-polyacrylamide gel ([C ₄ mim]BF ₄ -PAG), Using the gel, the protein molecular weight markers were separated by ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The method is shown in Figure 1.

The preparation method and electrophoresis method of the gel are as follows:

(1) Preparation of ionic liquid-polyacrylamide gel

The mass concentration of 1-ethyl-3-methylimidazolium tetrafluoroborate prepared respectively is 0.05% (w/v), 0.1% (w/v), 0.2% (w/v), 0.3% (w/v ), 0.6% (w/v) and 1.0% (w/v) 1-ethyl-3-methylimidazolium tetrafluoroborate-polyacrylamide gel ([C ₂ mim]BF ₄ -PAG) And 1-butyl-3-methylimidazolium tetrafluoroborate mass concentration is 0.05% (w/v), 0.1% (w/v), 0.2% (w/v), 0.3% (w/v) , 0.6% (w/v) and 1.0% (w/v) 1-butyl-3-methylimidazolium tetrafluoroborate-polyacrylamide gel ([C ₄ mim]BF ₄ -PAG), The mass concentration of acrylamide and N, N'-methylenebisacrylamide in the separating gel is 14% (w/v), and the mass concentration of acrylamide and N, N'-methylenebisacrylamide in the stacking gel 5.0% (w/v). Assemble the glue-making frame: use a 10×7.2×0.1cm glue-making glass plate, first wash and dry the two glass plates, and seal the two sides and the bottom of the glass plate with an appropriate amount of Vaseline to prevent leakage. Preparation of separation gel: add ionic liquid, 1.46g acrylamide, 40mg N,N'-methylene bisacrylamide and 454mg Tris-base to different 10mL volumetric flasks respectively, add water to 8mL, and use Adjust the pH value to 8.8 with 6mM HCl, and dilute to 10mL with water; mix well, transfer to a centrifuge tube, add 100μL of 10% ammonium persulfate prepared on the day and 5μL of N,N,N',N'-tetramethyl ethylenediamine, oscillating and mixing for 20s, after mixing evenly, ultrasonically degassed for 10s to obtain a separation gel solution; pour 5mL of the separation gel solution into a glass plate, seal and solidify with water, pour off the upper layer of water after the separation gel is solidified, and absorb it with filter paper Dry; prepare stacking gel: Add ionic liquid, 486mg acrylamide, 14mg N, N'-methylenebisacrylamide and 151mg Tris-base to different 10mL volumetric flasks respectively, add water to about 8mL , adjust the pH value to 6.8 with 6mM HCl, and dilute to 10mL with water; after mixing evenly, transfer to a centrifuge tube, add 50μL of 10% ammonium persulfate prepared on the day and 5μL of N, N, N', N'- Tetramethylethylenediamine, oscillating and mixing for 20s, after mixing evenly, ultrasonically degassed for 10s to obtain the concentrated gel solution, inject the concentrated gel solution into the glass plate, then insert the comb into the glass plate until the gel solidifies, pull out the comb, A sample injection hole is formed, and an ionic liquid-polyacrylamide gel is prepared for electrophoresis.

Table 1 adds the quality of ionic liquid

(2) Pretreatment of protein samples

The protein sample is a protein molecular weight marker with a molecular weight range of 99.0-14.4 KDa, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and no pretreatment is required before use. the

(3) Prepare protein electrophoresis buffer and load it

Add 3.03g of Tris-base, 14.4g of glycine, and 1g of sodium dodecyl sulfate to a 1L volumetric flask, and dilute to volume with water to prepare protein electrophoresis buffer; then assemble the electrophoresis tank, pour the electrophoresis buffer into the For the short plate of the plastic frame glass plate, inject 10 μL of the protein Maker sample into the injection hole using a micro-injector. In Figure 2, the mass concentrations of [C ₂ mim ]BF ₄ in the 1st to 6th channels are respectively: 0.05% ( w/v), 0.1% (w/v), 0.2% (w/v), 0.3% (w/v), 0.6% (w/v) and 1.0% (w/v); in Figure 3, the first The mass concentrations of [C ₄ mim ]BF ₄ in channels 1 to 6 are: 0.05% (w/v), 0.1% (w/v), 0.2% (w/v), 0.3% (w/v), 0.6% (w/v) and 1.0% (w/v).

(4) Electrophoresis

Switch on the electrophoresis apparatus. First use 120V voltage for electrophoresis until the bromophenol blue reaches the separation boundary of the separation gel and the stacking gel, then adjust the voltage to 160V until the bromophenol blue electrophoresis reaches the bottom of the gel. the

(5) Dyeing and decolorization

After the electrophoresis, the gel was peeled off the glass plate in water, and then washed twice with water for staining; staining was performed with 0.5% Coomassie Brilliant Blue R250 staining solution for 30 minutes, and then mixed with 20% methanol and 20% acetic acid The solution was decolorized until the background color was removed. the

The experimental results are shown in Figure 2 and Figure 3, Figure 2 is the mass concentration of [C ₂ mim]BF ₄ prepared in Example 1 is 0.05% (w/v), 0.1% (w/v), 0.2% (w /v), 0.3% (w/v), 0.6% (w/v) and 1.0% (w/v) [C ₂ mim]BF ₄ -PAG electrophoresis separation of protein molecular weight marker electrophoresis. Fig. 3 is [C ₄ mim] BF ₄ mass concentration that embodiment example 1 prepares is 0.05% (w/v), 0.1% (w/v), 0.2% (w/v), 0.3% (w/v) , 0.6% (w/v) and 1.0% (w/v) [C ₄ mim]BF ₄ -PAG) electrophoretic separation of protein molecular weight Marker electrophoresis. The results of electrophoresis showed that in the ionic liquid-polyacrylamide gel, when the mass concentration of the ionic liquid ranged from 0.05 to 1.0% (w/v), the protein molecular weight marker could be successfully separated. Increasing the content of ionic liquid in ionic liquid-polyacrylamide gel will significantly reduce the migration distance of proteins, and the different alkyl side chains of ionic liquid will also have different effects

Example 2

The brominated N-butyl-3-methylpyridine bromide mass concentration is 0.05% (w/v), 0.3% (w/v) and 1.0% (w/v) respectively The picoline-polyacrylamide gel ([C ₄ mpd]Br-PAG), the mass concentration of N-butyl-3-methylpyrrolidine bromide is 0.05% (w/v), 0.3% (w/v ) and 1.0% (w/v) of N-butyl-3-methylpyrrolidine bromide-polyacrylamide gel ([C ₄ mprd]Br-PAG) and 1-butyl-3-methyl bromide The bromide 1-butyl-3-methylimidazole-polyacrylamide gel ([C ₄ mim] Br-PAG), using the gel to carry out ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation on ten-fold diluted human serum samples, the method is shown in Figure 1.

(1) Preparation of ionic liquid-polyacrylamide gel

[C ₄ mpd]Br-PAG, [C 4 mpd]Br with mass concentrations of [C ₄ mpd]Br of 0.05% (w/v), _0.3 % (w/v) and 1.0% (w/v) were prepared respectively [C 4 mprd]Br _{-PAG with a mass concentration of 0.05% (w/v), 0.3% (w/v) and 1.0% (w/v) and [C 4 mim} ]Br with a mass concentration of 0.05% (w/ v), 0.3% (w/v) and 1.0% (w/v) [C ₄ mim]Br-PAG, wherein the mass concentration of acrylamide and N, N'-methylene in the separating gel is 12% ( w/v), the mass concentration of acrylamide and N,N'-methylenebisacrylamide in the stacking gel was 4.0% (w/v). Assemble the glue-making frame: use a 10×7.2×0.1cm glue-making glass plate, first wash and dry the two glass plates, and seal the sides and bottom of the glass plate with an appropriate amount of Vaseline to prevent leakage. Preparation of separation gel: add ionic liquid, 1.168g acrylamide, 32mg N,N'-methylenebisacrylamide and 454mg Tris-base to different 10mL volumetric flasks respectively, add water to 8mL, and use Adjust the pH value to 8.8 with 6mM HCl, and dilute to 10mL with water; mix well, transfer to a centrifuge tube, add 100μL of 10% ammonium persulfate prepared on the day and 5μL of N,N,N',N'-tetramethyl ethylenediamine, oscillating and mixing for 20s, and after mixing evenly, ultrasonic degassing for 10s to obtain a separation gel solution; pour 5mL of the separation gel solution into a glass plate, and seal it with water to solidify; after the separation gel solidifies, pour off the upper layer of water, and absorb Dry; prepare stacking gel: Add ionic liquid, 389mg acrylamide, 11mg N, N'-methylenebisacrylamide and 151mg Tris-base to different 10mL volumetric flasks respectively, add water to 8mL, Use 6mM HCl to adjust the pH to 6.8, and dilute to 10mL with water; after mixing evenly, transfer to a centrifuge tube, add 50μL of 10% ammonium persulfate prepared on the day and 5μL of N,N,N',N'-tetramethyl ethylenediamine, oscillating and mixing for 20s, after mixing evenly, ultrasonically degassed for 10s to obtain a concentrated gel solution, inject the concentrated gel solution into a glass plate, and then insert a comb into the glass plate until the gel is solidified, then pull out the comb to form a concentrated gel solution. Sample wells were prepared by ionic liquid-polyacrylamide gel for electrophoresis.

Table 2 adds the quality of ionic liquid

(2) Pretreatment of protein samples

Human serum was diluted ten times with water, and mixed with 2×SDS-sample buffer (purchased from Tiangen Biochemical Technology Co., Ltd.) at a volume ratio of 1:1. After mixing evenly, heated in a water bath at 95°C for 5 minutes, and cooled at room temperature to obtain protein sample. the

(3) Prepare protein electrophoresis buffer and load it

Add 3.03g of Tris-base, 14.4g of glycine, and 1g of sodium dodecyl sulfate to a 1L volumetric flask, and dilute to volume with water to prepare protein electrophoresis buffer; then assemble the electrophoresis tank, pour the electrophoresis buffer into the Glue the short plate of the glass plate, and inject 10 μL of the pretreated protein sample into the injection hole using a micro-sampler. the

(4) Electrophoresis

Switch on the electrophoresis apparatus. Firstly, electrophoresis with a voltage of 100V until the bromophenol blue reaches the separation boundary of the separation gel and the stacking gel, and then adjust the voltage to 140V until the bromophenol blue electrophoresis reaches the bottom of the gel. the

(5) Dyeing and decolorization

After the electrophoresis, the gel was peeled off the glass plate in water, and then washed twice with water for staining; staining was performed with 0.5% Coomassie Brilliant Blue R250 staining solution for 1 hour, and then mixed with 20% methanol and 20% acetic acid The solution was decolorized until the background color was removed. the

In addition, a parallel control experiment SDS-PAGE was carried out. the

The experimental results are shown in Figures 4, 5 and 6. Figure 4 shows the [C ₄ mpd]Br-PAG and [C ₄ mpd]Br mass concentration of [C ₄ mpd]Br prepared in Example 2 with a mass concentration of 0.05%. Electropherogram of 10-fold diluted human serum separated by 0.05% [C ₄ mprd]Br-PAG and [C ₄ mim]Br-PAG with a mass concentration of [C ₄ mim]Br-PAG of 0.05% and its parallel control experiment SDS-PAGE Electropherogram of separation of ten-fold dilutions of human serum. Figure 5 shows [C ₄ mpd]Br-PAG with [C ₄ mpd]Br mass concentration of 0.3% and [C ₄ mprd]Br-PAG with [C ₄ mpd]Br mass concentration of 0.3% prepared in Example 2 The electropherogram of the ten-fold dilution of human serum separated by [C ₄ mim]Br-PAG with a mass concentration of [C ₄ mim]Br of 0.3% and the electropherogram of the parallel control experiment SDS-PAGE of the ten-fold dilution of human serum. Figure 6 shows [C ₄ mpd]Br-PAG with [C ₄ mpd]Br mass concentration of 1.0% and [C ₄ mprd]Br-PAG with [C ₄ mprd]Br mass concentration of 1.0% prepared in Example 2 The electrophoresis of the ten-fold dilution of human serum separated by [C ₄ mim]Br-PAG with a mass concentration of [C ₄ mim]Br of 1.0% and the electrophoresis of the ten-fold dilution of human serum by SDS-PAGE in a parallel control experiment. The results of electrophoresis show that compared with the parallel control experiment SDS-PAGE, the ionic liquid-polyacrylamide gel prepared in this embodiment and using it to carry out ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis have a significant effect on human serum Proteins below 66.2KDa have obvious separation advantages. It can be seen from the figure that for human serum proteins with molecular weights ranging from 66.2 to 26.0 KDa, imidazoles and pyridines with a mass concentration range of 0.05 to 1.0% (w/v) of alkyl carbon chains with carbon atoms ≤ 4 are used. And pyrrolidine ionic liquid-polyacrylamide gel, the resolution and the number of bands are better than the parallel control experiment SDS-PAGE. Wherein, the degree of separation and the number of bands of the protein increase as the concentration of the ionic liquid increases. Taking [C ₄ mpd]Br-PAG as an example, as the concentration of ionic liquid increases, the number of clear protein bands in this region increases from 2 to 6. When 1.0% [C ₄ mpd]Br-PAG is used , the separated protein obtains the best number of separated bands and degree of separation. For human serum proteins with molecular weights ranging from 26.0 to 12.0 KDa, imidazoles, pyridines and pyrrolidines with a mass concentration range of 0.3 to 1.0% (w/v) of alkyl carbon chains and carbon atoms ≤ 4 ionic liquid-polymer Acrylamide gel is more suitable, and the resolution and the number of bands are better than the parallel control experiment SDS-PAGE. Among them, the degree of separation and the number of bands increased with the concentration of ionic liquids. As can be seen from Fig. 4, the low concentration of ionic liquid (0.05%) can not improve the degree of separation of proteins in this molecular weight range and increase the number of separated bands; when Fig. 5 uses 0.3% ionic liquid-polyacrylamide gel, the The number of protein bands in the area increased from 1 to 2. When using 1.0% ionic liquid-polyacrylamide gel, the number of protein bands in this area increased from 2 to 3. At this time, the separated proteins obtained the most The number and resolution of the best separated bands.

The present invention includes but is not limited to the above embodiments, and any equivalent replacement or partial improvement made under the spirit and principle of the present invention will be considered within the protection scope of the present invention. the

Claims (5)

1. the purposes of an ionic liquid-polyacrylamide gel, this gel mainly comprises monomer, linking agent, gel buffer system, catalyzer and accelerator, also comprises ionic liquid; Wherein, described ionic liquid is the ionic liquid of alkyl carbon chain carbonatoms≤4 on positively charged ion, and the mass concentration of described ionic liquid is 0.05～1.0%(w/v); On described positively charged ion the ionic liquid of alkyl carbon chain carbonatoms≤4 be alkyl carbon chain carbonatoms≤4 on positively charged ion glyoxaline ion liquid, pyridines ionic liquid or pyrrolidines ionic liquid one of them; When the mass concentration of described ionic liquid is 0.3～1.0%(w/v) for separating of the protein of molecular weight ranges at 26.0～12.0KDa; The preparation method of described ionic liquid-polyacrylamide gel is for first being mixed to get mixing solutions with ionic liquid, monomer, linking agent and gel buffer system, add described mixing solutions to mix catalyzer and accelerator again, after solidifying, can obtain described a kind of ionic liquid-polyacrylamide gel; It is characterized in that: described a kind of ionic liquid-polyacrylamide gel purposes is that ionic liquid-polyacrylamide gel is used for electrophoretic analysis protein, be ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, described electrophoresis method step is as follows:

(1) prepare a kind of ionic liquid-polyacrylamide gel;

(2) SDS pre-treatment protein example;

(3) preparation protein electrophorese damping fluid and loading;

(4) electrophoresis;

(5) dyeing and decolouring;

Specifically, described electrophoresis method mainly comprises:

(1) preparation ionic liquid-polyacrylamide gel

Assembling sheet glass gum-making rack, first ionic liquid, monomer, linking agent and gel buffer system are mixed to get mixing solutions, after adding described mixing solutions to mix catalyzer and accelerator again ionic liquid-polyacrylamide gel solution, described gelating soln is injected the sheet glass of glue frame, afterwards comb is inserted in the gelating soln in sheet glass, until gel solidifies, comb is extracted, form sample holes;

(2) pre-treatment protein example

Add sample buffered soln in protein example, mix post-heating, then cooling, obtain the pre-treatment protein example;

(3) preparation protein electrophorese damping fluid and loading

The assembling electrophoresis apparatus, preparation protein electrophorese damping fluid also adds in electrophoresis chamber; Pour the short slab that the protein electrophorese damping fluid did not have glue frame sheet glass into, utilize microsyringe with pre-treatment after protein example injector tunnel loading;

(4) electrophoresis

Connect electrophoresis apparatus and carry out electrophoresis;

(5) dyeing and decolouring

Electrophoresis peels gel after finishing from sheet glass, water cleans, and dyes, and the processing of decolouring after dyeing is completed is until remove the background background color.

2. the purposes of a kind of ionic liquid-polyacrylamide gel according to claim 1, is characterized in that: the both sides and the bottom that seal the gum-making rack sheet glass when assembling gum-making rack in step (1) with Vaseline.

3. the purposes of a kind of ionic liquid-polyacrylamide gel according to claim 1 is characterized in that: add sample buffered soln in protein example in step (2), and after mixing, at 95 ℃ of heating in water bath 5min, cooling under room temperature; The mass concentration of protein example is 0.1～0.5mg/mL.

4. the purposes of a kind of ionic liquid-polyacrylamide gel according to claim 1 is characterized in that: in step (3), the loading volume of protein example is 5～10 μ L.

5. the purposes of a kind of ionic liquid-polyacrylamide gel according to claim 1, is characterized in that: after in step (5), electrophoresis finishes, in water, gel is peeled from sheet glass.

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