CN102229688A - Ionic liquid- polyacrylamide gel, preparation method and purpose thereof - Google Patents
Ionic liquid- polyacrylamide gel, preparation method and purpose thereof Download PDFInfo
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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
Technical field
The present invention relates to a kind of ionic liquid-polyacrylamide gel (ILs-PAG), its method for making and purposes, specifically, ionic liquid in described ionic liquid-polyacrylamide gel (ILs) is the ionic liquid of alkyl carbon chain carbonatoms≤4 on the positively charged ion, belongs to the protein separation analysis technical field.
Background technology
After the human genome order-checking was finished, proteomics research had obtained paying close attention to widely.In recent years, proteomics research was widely used in fields such as basic life science, medical analysis and disease marker research.Complete proteome analysis comprises specimen preparation, protein separation, protein analysis evaluation and characterizes four parts that wherein protein separation and analytical technology are the keys of proteomics research, are restricting the development of proteomics research.
Propose sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) from shapizo in 1967, SDS-PAGE is widely used in bioseparation and proteomics field as a kind of protein separation analytical technology of classics.The ultimate principle of SDS-PAGE is that protein combines the electronegative protein-SDS mixture of formation with sodium lauryl sulphate (SDS), can eliminate the charge differences between the different proteins molecule, thereby make the electrophoretic mobility of protein molecule in polyacrylamide gel (PAG) no longer be subjected to the influence of the original electric charge of protein, and depending primarily on the size of protein molecule quality, the logarithm of its mobility and protein molecular weight is linear.This character of SDS-PAGE can realize proteinic effective separation, and can utilize the relation between migration distance and the protein molecular weight that protein is carried out qualitative examination.But, equate or close different proteins that for molecular weight SDS-PAGE can not be to existing effectively separation and evaluation in fact.
Ionic liquid claims ionic liquid at room temperature or room temperature melting salt again, is meant in room temperature or near presenting the liquid salt that melts under the room temperature, is made of organic cation and inorganic anion usually.Ionic liquid not only has the advantage of conventional organic solvents, and has the advantage of many uniquenesses, as steam at ambient temperature force down, not flammable, good stability, dissolving power is strong and electroconductibility is high.The most important thing is that ionic liquid can become a kind of programmable solvent by changing positively charged ion and the ion liquid physico-chemical property of anionic kind directed change.Have the functional group of special role by introducing some on the positively charged ion in ionic liquid, can also design ionic liquid with specific function.Just because of this, in recent decades, ionic liquid is widely used in the bioseparation analysis field.
Have and successfully utilize ionic liquid to improve the methods involving report of protein separation analytical technology.For example, article " Simultaneous separation of basic and acidic proteins using 1-butyl-3-methylimidazoliumbased ion liquid as dynamic coating and background electrolyte in capillary electrophoresis " (DOI 10.1002/elps.200700746,2008,29,2356～2362) method of Jie Shaoing, utilize 1-butyl-3-methyl imidazolium tetrafluoroborate as the electrolytical additive of capillary electrophoresis, five kinds of protein of different nature of successful baseline separation in 14 minutes.In this method, ionic liquid can dynamically be coated in the capillary tube inner wall surface, changes the charge property of inwall, thereby reduces the adsorption of capillary tube inner wall to basic protein.Up to the present, the method that is applied to improve SDS-PAGE and other gel separation technology about ionic liquid is report not also, and the present invention has just in time solved this problem.
Summary of the invention
At the SDS-PAGE method molecular weight is equated or the close low defective of different proteins resolution that one of purpose of the present invention is to provide a kind of ionic liquid-polyacrylamide gel; Specifically, the ionic liquid in described ionic liquid-polyacrylamide gel is the ionic liquid of alkyl carbon chain carbonatoms≤4 on the positively charged ion.
Two of purpose of the present invention is to provide a kind of preparation method of ionic liquid-polyacrylamide gel.
Three of purpose of the present invention is to provide a kind of purposes of ionic liquid-polyacrylamide gel, described purposes is that ionic liquid-polyacrylamide gel is used for electrophoretic analysis protein, i.e. ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (ILs-SDS-PAGE).Use the protein of described electrophoresis method electrophoretic separation after SDS handles, molecular weight is equated or close different proteins can be realized quick, easy, green and separation efficiently.
For realizing purpose of the present invention, the technical scheme of employing is as follows.
A kind of ionic liquid-polyacrylamide gel, described gel is made of jointly ionic liquid and polyacrylamide gel prescription, it is a kind of ionic liquid-polyacrylamide gel, mainly comprise monomer, linking agent, gel buffer system, catalyzer and accelerator, it is characterized in that: also comprise ionic liquid.
Wherein, described ionic liquid is the ionic liquid of alkyl carbon chain carbonatoms≤4 on the positively charged ion, includes but not limited to glyoxaline ion liquid, pyridines ionic liquid or the pyrrolidines ionic liquid of alkyl carbon chain carbonatoms≤4.
Described ion liquid mass concentration scope is 0.05～1.0% (w/v).
Described polyacrylamide gel prescription is this area general polyacrylamide gel prescription in protein separation is analyzed, and includes but not limited to following component: monomer, linking agent, gel buffer system, catalyzer and accelerator; Wherein, each component includes but not limited to: monomer acrylamide, linking agent N, N '-methylene-bisacrylamide, gel buffer system, catalyzer ammonium persulphate (APS) and accelerator N,N,N (TEMED).A kind of in the wherein said gel buffer system polyacrylamide gel buffer system that to be this area in protein separation is analyzed general includes but not limited to Tris-HCl buffer system, borax-borate buffer system or Sodium phosphate dibasic and SODIUM PHOSPHATE, MONOBASIC buffer system.The mass concentration of each component is all this area general polyacrylamide gel in the protein separation analysis each constituent mass concentration of filling a prescription, and determines according to actual needs.
The gelling system of ionic liquid-polyacrylamide gel of the present invention is continuous gelling system or discontinuous gelling system, and is identical with this area general polyacrylamide gel system in protein separation is analyzed.Include but not limited to acrylamide and N in the polyacrylamide gel of continuous gelling system, N '-methylene-bisacrylamide mass concentration is 4～17% (w/v), and the pH value is 5.8～8.8; Acrylamide and N in the discontinuous gelling system in separation gel and the concentrated glue, N '-methylene-bisacrylamide mass concentration is 4～17% (w/v), and wherein resolving gel concentration is greater than concentrated gum concentration, and the pH value is 5.8～8.8.
Described ion liquid mass concentration is according to the main molecules amount and the gel strength decision of the isolating target protein matter of need.Wherein, described protein is this area protein that SDS-PAGE was suitable in protein separation is analyzed, and includes but not limited to that protein molecular weight Marker and molecular weight are lower than the human serum protein biological sample of 66.2KDa.
For protein molecular weight Marker, using mass concentration scope provided by the invention is the ionic liquid-polyacrylamide gel of glyoxaline ion liquid, pyridines ionic liquid or the preparation of pyrrolidines ionic liquid of alkyl carbon chain carbonatoms≤4 of 0.05～1.0% (w/v), can successfully separate.
For the protein of molecular weight ranges among the human serum sample at 66.2～26.0KDa, using mass concentration scope provided by the invention is glyoxaline ion liquid, pyridines ionic liquid or pyrrolidines ionic liquid-polyacrylamide gel separation of alkyl carbon chain carbonatoms≤4 of 0.05～1.0% (w/v), control experiment SDS-PAGE in parallel relatively, improve proteinic resolution, increased the clear protein band number of molecular weight phase near field.Wherein, described proteinic resolution and band number increase and increase along with ionic liquid concentration.Preferred functional quality concentration is pyridines ionic liquid-polyacrylamide gel of 1.0% (w/v), and this moment, isolating protein obtained best separation band number and resolution.
For the protein of molecular weight ranges among the human serum sample at 26.0～12.0KDa, the functional quality concentration range is that imidazoles, pyridines and the pyrrolidines ionic liquid-polyacrylamide gel of alkyl carbon chain carbonatoms≤4 of 0.3～1.0% (w/v) separates, control experiment SDS-PAGE in parallel relatively, improve proteinic resolution, can increase the clear protein band number of molecular weight phase near field.Wherein, protein separation degree and band number increase and increase along with ionic liquid concentration.Preferred functional quality concentration is pyridines ionic liquid-polyacrylamide gel of 1.0% (w/v), and this moment, institute's isolated protein obtained best separation band number and resolution.
A kind of preparation method of ionic liquid-polyacrylamide gel, described method steps is as follows:
Earlier ionic liquid, monomer, linking agent and gel buffer system are mixed obtaining mixing solutions, again catalyzer and accelerator are added described mixing solutions and mix, after solidifying, can obtain a kind of ionic liquid-polyacrylamide gel of the present invention.
Wherein, described ionic liquid is the ionic liquid of alkyl carbon chain carbonatoms≤4 on the positively charged ion, includes but not limited to glyoxaline ion liquid, pyridines ionic liquid or the pyrrolidines ionic liquid of alkyl carbon chain carbonatoms≤4.
Described ion liquid mass concentration scope is 0.05～1.0% (w/v).
Outside the deionization liquid, other preparation methods of ionic liquid-polyacrylamide gel of the present invention are this area general polyacrylamide gel preparation method in protein separation is analyzed.Include but not limited to earlier ionic liquid, monomer, linking agent and the gel buffer system of suitable proportion be mixed obtain mixing solutions, again catalyzer and accelerator being added described mixing solutions mixes, after solidifying, can obtain a kind of ionic liquid-polyacrylamide gel of the present invention.Include but not limited to ionic liquid, monomer acrylamide, the linking agent N of elder generation with suitable proportion, N '-methylene-bisacrylamide and the mixing of gel buffer system obtain mixing solutions, again with catalyzer ammonium persulphate and accelerator N, N, N ', N '-Tetramethyl Ethylene Diamine adds described mixing solutions and mixes, and after solidifying, can obtain a kind of ionic liquid-polyacrylamide gel of the present invention.A kind of in the wherein said gel buffer system polyacrylamide gel buffer system that to be this area in protein separation is analyzed general includes but not limited to Tris-HCl buffer system, borax-borate buffer system or Sodium phosphate dibasic and SODIUM PHOSPHATE, MONOBASIC buffer system.Wherein the mass concentration of monomer, linking agent and gel buffer system, catalyzer and accelerator is all this area general polyacrylamide gel in the protein separation analysis each constituent mass concentration of filling a prescription, and determines according to actual needs.
The gelling system of ionic liquid-polyacrylamide gel of the present invention is continuous gelling system or discontinuous gelling system, and is identical with this area general polyacrylamide gel system in protein separation is analyzed.Include but not limited to acrylamide and N in the polyacrylamide gel of continuous gelling system, the mass concentration of N '-methylene-bisacrylamide is 4～17% (w/v), and the pH value is 5.8～8.8; Acrylamide and N in the discontinuous gelling system in separation gel and the concentrated glue, N '-methylene-bisacrylamide mass concentration is 4～17% (w/v), and wherein resolving gel concentration is greater than concentrated gum concentration, and the pH value is 5.8～8.8.
Described ion liquid mass concentration is according to the main molecules amount and the gel strength decision of the isolating target protein matter of need.Wherein, described protein is this area protein that SDS-PAGE was suitable in protein separation is analyzed, and includes but not limited to that protein molecular weight Marker and molecular weight are lower than the human serum protein biological sample of 66.2KDa.
For protein molecular weight Marker, using mass concentration scope provided by the invention is the ionic liquid-polyacrylamide gel of glyoxaline ion liquid, pyridines ionic liquid or the preparation of pyrrolidines ionic liquid of alkyl carbon chain carbonatoms≤4 of 0.05～1.0% (w/v), can successfully separate.
For the protein of molecular weight ranges among the human serum sample at 66.2～26.0KDa, using mass concentration scope provided by the invention is glyoxaline ion liquid, pyridines ionic liquid or pyrrolidines ionic liquid-polyacrylamide gel separation of alkyl carbon chain carbonatoms≤4 of 0.05～1.0% (w/v), control experiment SDS-PAGE in parallel relatively, improve proteinic resolution, increased the clear protein band number of molecular weight phase near field.Wherein, described proteinic resolution and band number increase and increase along with ionic liquid concentration.Preferred functional quality concentration is pyridines ionic liquid-polyacrylamide gel of 1.0% (w/v), and this moment, isolating protein obtained best separation band number and resolution.
For the protein of molecular weight ranges among the human serum sample at 26.0～12.0KDa, the functional quality concentration range is that imidazoles, pyridines and the pyrrolidines ionic liquid-polyacrylamide gel of alkyl carbon chain carbonatoms≤4 of 0.3～1.0% (w/v) separates, control experiment SDS-PAGE in parallel relatively, improve proteinic resolution, can increase the clear protein band number of molecular weight phase near field.Wherein, protein separation degree and band number increase and increase along with ionic liquid concentration.Preferred functional quality concentration is pyridines ionic liquid-polyacrylamide gel of 1.0% (w/v), and this moment, institute's isolated protein obtained best separation band number and resolution.
A kind of purposes of ionic liquid-polyacrylamide gel, described purposes are that ionic liquid-polyacrylamide gel is used for electrophoretic analysis protein, i.e. ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Described electrophoresis method step is as follows:
(1) prepares a kind of ionic liquid-polyacrylamide gel;
(2) SDS pre-treatment protein example;
(3) preparation protein electrophorese damping fluid and upward sample;
(4) electrophoresis;
(5) dyeing and decolouring.
It is levied and is: the employed gel of described electrophoresis is ionic liquid-polyacrylamide gel of the present invention, and all the other deposition conditions are with this area deposition condition that SDS-PAGE was suitable in protein separation is analyzed.
Specifically, described electrophoresis method includes but not limited to:
(1) preparation ionic liquid-polyacrylamide gel
Assembling sheet glass gum-making rack preferably with the both sides and the bottom of Vaseline sealing gum-making rack sheet glass, prevents leakage; If continuous gelling system, earlier ionic liquid, monomer, linking agent and gel buffer system are mixed and obtain mixing solutions, again catalyzer and accelerator are added described mixing solutions after mixing 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 the sheet glass, solidify until gel, comb is extracted, form sample holes; If discontinuous gelling system need be made separation gel and concentrated glue respectively, method is similar with continuous gel colloid.
(2) pre-treatment protein example
Add sample buffered soln in protein example, mix post-heating, cooling obtains the pre-treatment protein example then.Wherein, preferably, cool off under the room temperature at 95 ℃ of heating in water bath 5min; The mass concentration of protein example is preferably 0.1～0.5mg/mL; Sample buffer includes but not limited to buffer system, SDS, glycerine, tetrabromophenol sulfonphthalein, beta-mercaptoethanol and water, the mass ratio of SDS and protein example 〉=1.45: 1; In the gelling system, the kind of sample buffer buffer system, concentration and pH value are identical with the gel buffer system of ionic liquid-polyacrylamide gel system continuously; In discontinuous gelling system, the kind of sample buffer buffer system, concentration and pH value are identical with the gel buffer system that ionic liquid-polyacrylamide gel system concentrates sol solution.
(3) preparation protein electrophorese damping fluid and upward sample
The assembling electrophoresis apparatus, preparation protein electrophorese damping fluid also adds in the electrophoresis chamber; Pour the short slab that the protein electrophorese damping fluid did not have glue frame sheet glass into, utilize microsyringe will be on the pretreated protein example of step (2) injector tunnel sample.
Wherein, the last sample volume of protein example is preferably 5～10 μ L.In the gelling system, the kind of electrophoretic buffer buffer system, concentration and pH value are identical with the gel buffer system of ionic liquid-polyacrylamide gel system continuously; In discontinuous gelling system, the kind of electrophoretic buffer buffer system, concentration and pH value are identical with the gel buffer system that ionic liquid-polyacrylamide gel system concentrates sol solution.
(4) electrophoresis
Connect electrophoresis apparatus and carry out electrophoresis.Gelling system adopts the constant voltage electrophoresis continuously.Discontinuous gelling system at first concentrates with low voltage, when the stall limit of tetrabromophenol sulfonphthalein arrival separation gel and concentrated glue, uses high relatively voltage instead and carries out electrophoretic separation, arrives the gel bottom until tetrabromophenol sulfonphthalein, and electrophoresis finishes.
Wherein, in the gelling system, voltage is 80～200V continuously; In the discontinuous gelling system, the voltage that concentrates glue is generally 80～140V, and the voltage of separation gel is 100～160V.
(5) dyeing and decolouring
Electrophoresis peels gel after finishing from sheet glass, water dyes after cleaning, and the processing of decolouring after dyeing is finished is until removing the background background color.
Preferably in water, gel is peeled from sheet glass, break to prevent gel.
Wherein dyeing includes but not limited to coomassie brilliant blue R250 staining, Coomassie brilliant blue G250 staining or argentation for the staining technique that this area is suitable in protein separation is analyzed.
Described ion liquid mass concentration is according to the main molecules amount and the gel strength decision of the isolating target protein matter of need.Wherein, described protein is this area protein that SDS-PAGE was suitable in protein separation is analyzed, and includes but not limited to that protein molecular weight Marker and molecular weight are lower than the human serum protein biological sample of 66.2KDa.
For protein molecular weight Marker, using mass concentration scope provided by the invention is that the glyoxaline ion liquid, pyridines ionic liquid of alkyl carbon chain carbonatoms≤4 of 0.05～1.0% (w/v) or the ionic liquid-polyacrylamide gel of pyrrolidines ionic liquid preparation carry out ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, can successfully separate.
For the protein of molecular weight ranges among the human serum sample at 66.2～26.0KDa, using mass concentration scope provided by the invention is that glyoxaline ion liquid, pyridines ionic liquid or the pyrrolidines ionic liquid-polyacrylamide gel of alkyl carbon chain carbonatoms≤4 of 0.05～1.0% (w/v) carries out ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, control experiment SDS-PAGE in parallel relatively, improve proteinic resolution, increased the clear protein band number of molecular weight phase near field.Wherein, described proteinic resolution and band number increase and increase along with ionic liquid concentration.Preferred functional quality concentration is pyridines ionic liquid-polyacrylamide gel of 1.0% (w/v), and this moment, isolating protein obtained best separation band number and resolution.
For the protein of molecular weight ranges among the human serum sample at 26.0～12.0KDa, the functional quality concentration range is that imidazoles, pyridines and the pyrrolidines ionic liquid-polyacrylamide gel of alkyl carbon chain carbonatoms≤4 of 0.3～1.0% (w/v) carries out ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, control experiment SDS-PAGE in parallel relatively, improve proteinic resolution, can increase the clear protein band number of molecular weight phase near field.Wherein, protein separation degree and band number increase and increase along with ionic liquid concentration.Preferred functional quality concentration is pyridines ionic liquid-polyacrylamide gel of 1.0% (w/v), and the isolating protein of institute's this moment obtains the separation band number and the resolution of the best.
Beneficial effect
1. efficient and favorable reproducibility simply, fast.The present invention adds the ionic liquid of alkyl carbon chain carbonatoms≤4 on the positively charged ion on polyacrylamide gel prescription basis, prepare a kind of ionic liquid-polyacrylamide gel; On the basis of SDS-PAGE method, utilize ionic liquid that the method for SDS-PAGE is improved, use ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis to come isolated protein, have the advantage of simple, quick, efficient and favorable reproducibility.SDS-PAGE is classical protein separation analytical technology, and the method maturation is simple to operation, is widely used in the every field of protein research.Utilize ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ionic liquid improvement SDS-PAGE simple to operation equally, and with respect to SDS-PAGE, ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis isolated protein has higher resolution;
2. the glue process is difficult for producing bubble.Because the ionic liquid-polyacrylamide gel among the present invention does not add SDS, reduced the bubble that forms because of SDS, conveniently gelating soln is transferred in the sheet glass of glue frame, also reduced the generation of bubble in the gel process of setting, make the electrophoresis environment more stable;
3. to proteinic selective separation.SDS-PAGE can not realize effective separation for the different proteins of equal or close molecular weight.Utilize ionic liquid-polyacrylamide gel provided by the invention and since in ionic liquid-polyacrylamide gel the ionic liquid that has can with protein interaction, thereby influence proteinic rate of migration, realize the close proteinic separation of molecular weight; Adopt ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis provided by the invention, can realize that for equating or the better selective separation of the different proteins of close molecular weight especially the human serum protein that molecular weight is lower than 66.2KDa has selective separation efficiently.
4. security.The ionic liquid safety non-pollution is called as green solvent, can not produce harm to human body and environment;
5. universality.Ionic liquid-polyacrylamide gel provided by the invention and corresponding ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis method of setting up, can directly apply to protein separation, also be applicable to the improvement of polyacrylamide gel electrophoresis method, be expected to become the important technology of proteomics and protein separation analysis;
6. prevent that gel from breaking.In ionic liquid provided by the invention-sodium dodecyl sulfate-polyacrylamide gel electrophoresis method steps (5), in water, ionic liquid-polyacrylamide gel is peeled from sheet glass, can prevent that gel from breaking;
7. prevent leakage.When assembling gum-making rack in ionic liquid provided by the invention-sodium dodecyl sulfate-polyacrylamide gel electrophoresis method steps (1), both sides and bottom with Vaseline sealing gum-making rack sheet glass can prevent leakage.
Description of drawings
Fig. 1 is ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis method of separating protein synoptic diagram.
The 1-ethyl that Fig. 2 prepares for embodiment 1-3-methyl imidazolium tetrafluoroborate mass concentration is 1-ethyl-3-methyl imidazolium tetrafluoroborate-polyacrylamide gel ([C of 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)
2Mim] BF
4-PAG) the electrophorogram of electrophoretic separation protein molecular weight Marker.
The 1-butyl that Fig. 3 prepares for embodiment 1-3-methyl imidazolium tetrafluoroborate mass concentration is 1-butyl-3-methyl imidazolium tetrafluoroborate-polyacrylamide gel ([C of 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)
4Mim] BF
4-PAG) the electrophorogram of electrophoretic separation protein molecular weight Marker.
The bromination N-butyl that Fig. 4 prepares for embodiment 2-3-picoline mass concentration is bromination N-butyl-3-picoline-polyacrylamide gel ([C of 0.05% (w/v)
4Mpd] Br-PAG), bromination N-butyl-3-crassitude mass concentration is bromination N-butyl-3-methylpyrrolidin-polyacrylamide gel ([C of 0.05% (w/v)
4Mprd] Br-PAG) and bromination 1-butyl-3-Methylimidazole mass concentration be bromination 1-butyl-3-Methylimidazole-polyacrylamide gel ([C of 0.05% (w/v)
4Mim] Br-PAG) electrophorogram and its parallel control of separating the human serum of ten times of dilutions test the electrophorogram that SDS-PAGE separates the human serum of ten times of dilutions.
The bromination N-butyl that Fig. 5 prepares for embodiment 2-3-picoline mass concentration is bromination N-butyl-3-picoline-polyacrylamide gel ([C of 0.3%
4Mpd] Br-PAG), bromination N-butyl-3-crassitude mass concentration is bromination N-butyl-3-methylpyrrolidin-polyacrylamide gel ([C of 0.3%
4Mprd] Br-PAG) and bromination 1-butyl-3-Methylimidazole mass concentration be bromination 1-butyl-3-Methylimidazole-polyacrylamide gel ([C of 0.3%
4Mim] Br-PAG) electrophorograms and its parallel controls experiment SDS-PAGE that separate ten times of dilution human serums separate ten times of electrophorograms that dilute human serums.
The bromination N-butyl that Fig. 6 prepares for embodiment 2-3-picoline mass concentration is bromination N-butyl-3-picoline-polyacrylamide gel ([C of 1.0%
4Mpd] Br-PAG), bromination N-butyl-3-crassitude mass concentration is bromination N-butyl-3-methylpyrrolidin-polyacrylamide gel ([C of 1.0%
4Mprd] Br-PAG) and bromination 1-butyl-3-Methylimidazole mass concentration be bromination 1-butyl-3-Methylimidazole-polyacrylamide gel ([C of 1.0%
4Mim] Br-PAG) electrophorograms and its parallel controls experiment SDS-PAGE that separate ten times of dilution human serums separate ten times of electrophorograms that dilute human serums.
Embodiment
In order to prove absolutely characteristic of the present invention and to implement mode of the present invention, provide embodiment below.
Prepare the 1-ethyl that 1-ethyl-3-methyl imidazolium 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)-3-methyl imidazolium tetrafluoroborate-polyacrylamide gel ([C respectively
2Mim] BF
4-PAG) and 1-butyl-3-methyl imidazolium tetrafluoroborate mass concentration be 1-butyl-3-methyl imidazolium tetrafluoroborate-polyacrylamide gel ([C of 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)
4Mim] BF
4-PAG), use described gel respectively protein molecular weight Marker to be carried out ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and separate.Method as shown in Figure 1.
The preparation method and the electrophoresis method of described gel are as follows:
(1) preparation ionic liquid-polyacrylamide gel
Prepare the 1-ethyl that 1-ethyl-3-methyl imidazolium 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)-3-methyl imidazolium tetrafluoroborate-polyacrylamide gel ([C respectively
2Mim] BF
4-PAG) and 1-butyl-3-methyl imidazolium tetrafluoroborate mass concentration be 1-butyl-3-methyl imidazolium tetrafluoroborate-polyacrylamide gel ([C of 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)
4Mim] BF
4-PAG), and wherein acrylamide and N in the separation gel, the mass concentration of N '-methylene-bisacrylamide is 14% (w/v), concentrates acrylamide and N in the glue, the mass concentration of N '-methylene-bisacrylamide is 5.0% (w/v).The assembling gum-making rack: use the glue sheet glass of 10 * 7.2 * 0.1cm, at first with two clean drying up of sheet glass, both sides and bottom with an amount of Vaseline sealing glass plate prevent leakage.Preparation separation gel: the ionic liquid, 1.46g acrylamide, the 40mg N that add quality as shown in table 1 in the different 10mL volumetric flasks respectively, N '-methylene-bisacrylamide and 454mg Tris-base, add water to 8mL, with the HCl adjust pH to 8.8 of 6mM, water is settled to 10mL; After mixing, be transferred in the centrifuge tube, add 10% ammonium persulphate that 100 μ L prepared the same day and the N,N,N of 5 μ L, vibration mixes 20s, mixes back ultrasonic degas 10s, obtains separation gel solution; 5mL separation gel solution is injected sheet glass, and water seal is solidified, and after separation gel solidifies by the time, upper water is outwelled, and blotted with filter paper; Preparation concentrates glue: the ionic liquid, 486mg acrylamide, the 14mgN that add quality as shown in table 1 in different 10mL volumetric flasks respectively, N '-methylene-bisacrylamide and 151mg Tris-base, add water to about 8mL, with the HCl adjust pH to 6.8 of 6mM, water is settled to 10mL; After mixing, be transferred in the centrifuge tube, add 10% ammonium persulphate that 50 μ L prepared the same day and the N of 5 μ L, N, N ', N '-Tetramethyl Ethylene Diamine, vibration mixes 20s, mixes back ultrasonic degas 10s, obtains concentrating sol solution, to concentrate sol solution and inject sheet glass, afterwards comb is inserted in the sheet glass, solidify, comb is extracted until gel, form sample holes, electrophoresis is finished with ionic liquid-polyacrylamide gel preparation.
Table 1 adds ion liquid quality
(2) pre-treatment protein example
Protein example is the protein molecular weight Marker of molecular weight ranges at 99.0～14.4KDa, available from TIANGEN Biotech (Beijing) Co., Ltd., need not pre-treatment before the use.
(3) preparation protein electrophorese damping fluid and upward sample
Add Tris-base, 14.4g glycine and the 1g sodium lauryl sulphate of 3.03g in the 1L volumetric flask, the water constant volume prepares the protein electrophorese damping fluid; Assemble electrophoresis chamber then, the electrophoretic buffer of pouring into did not have the short slab of gum-making rack sheet glass, utilized microsyringe that 10 μ L protein Maker samples are injected sample holes respectively, among Fig. 2, and [the C in the 1st～6 road
2Mim] BF
4Mass concentration be 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); Among Fig. 3, [the C in the 1st～6 road
4Mim] BF
4Mass concentration be 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).
(4) electrophoresis
Connect electrophoresis apparatus.At first use the voltage electrophoresis of 120V, to the stall limit of tetrabromophenol sulfonphthalein arrival separation gel and concentrated glue, regulating voltage arrives the gel bottom to 160V until the tetrabromophenol sulfonphthalein electrophoresis then.
(5) dyeing and decolouring
Electrophoresis peels gel in water respectively after finishing from sheet glass, water cleans twice afterwards, dyes; Use 0.5% coomassie brilliant blue R250 staining fluid dyeing 30min during dyeing, use 20% methyl alcohol and 20% acetic acid mixed solution to decolour then, until removing the background background color.
Experimental result as shown in Figures 2 and 3, [the C that Fig. 2 prepares for embodiment 1
2Mim] BF
4Mass concentration is the [C of 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)
2Mim] BF
4The electrophorogram of-PAG electrophoretic separation protein molecular weight Marker.[the C that Fig. 3 prepares for embodiment 1
4Mim] BF
4Mass concentration is the [C of 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)
4Mim] BF
4-PAG) the electrophorogram of electrophoretic separation protein molecular weight Marker.The electrophoresis result demonstration, in ionic liquid-polyacrylamide gel, when ionic liquid mass concentration scope is 0.05～1.0% (w/v), can successful isolated protein molecular weight Marker.The content that increases ionic liquid-polyacrylamide gel intermediate ion liquid can obviously reduce the protein migration distance, and the difference of ionic liquid alkyl group side chain also can produce different influences
Prepare the bromination N-butyl that bromination N-butyl-3-picoline mass concentration is 0.05% (w/v), 0.3% (w/v) and 1.0% (w/v)-3-picoline-polyacrylamide gel ([C respectively
4Mpd] Br-PAG), bromination N-butyl-3-crassitude mass concentration is bromination N-butyl-3-methylpyrrolidin-polyacrylamide gel ([C of 0.05% (w/v), 0.3% (w/v) and 1.0% (w/v)
4Mprd] Br-PAG) and bromination 1-butyl-3-Methylimidazole mass concentration be bromination 1-butyl-3-Methylimidazole-polyacrylamide gel ([C of 0.05% (w/v), 0.3% (w/v) and 1.0% (w/v)
4Mim] Br-PAG), use described gel respectively the human serum sample of ten times of dilutions to be carried out ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation, method is as shown in Figure 1.
(1) preparation ionic liquid-polyacrylamide gel
Prepare [C respectively
4Mpd] the Br mass concentration is the [C of 0.05% (w/v), 0.3% (w/v) and 1.0% (w/v)
4Mpd] Br-PAG, [C
4Mprd] the Br mass concentration is the [C of 0.05% (w/v), 0.3% (w/v) and 1.0% (w/v)
4Mprd] Br-PAG and [C
4Mim] the Br mass concentration is the [C of 0.05% (w/v), 0.3% (w/v) and 1.0% (w/v)
4Mim] Br-PAG, wherein acrylamide and N in the separation gel, the mass concentration of N '-methylene radical is 12% (w/v), concentrates acrylamide and N in the glue, the mass concentration of N '-methylene-bisacrylamide is 4.0% (w/v).The assembling gum-making rack: use the glue sheet glass of 10 * 7.2 * 0.1cm, at first with two clean drying up of sheet glass, both sides and bottom with an amount of Vaseline sealing glass plate prevent leakage.Preparation separation gel: the ionic liquid, 1.168g acrylamide, the 32mg N that add quality as shown in table 2 in the different 10mL volumetric flasks respectively, N '-methylene-bisacrylamide and 454mg Tris-base, add water to 8mL, with the HCl adjust pH to 8.8 of 6mM, water is settled to 10mL; After mixing, be transferred in the centrifuge tube, add 10% ammonium persulphate that 100 μ L prepared the same day and the N,N,N of 5 μ L, vibration mixes 20s, mixes back ultrasonic degas 10s, obtains separation gel solution; 5mL separation gel solution is injected sheet glass, and water seal is solidified; By the time after separation gel solidifies, upper water is outwelled, and blotted with filter paper; Preparation concentrates glue: the ionic liquid, 389mg acrylamide, the 11mgN that add quality as shown in table 2 in different 10mL volumetric flasks respectively, N '-methylene-bisacrylamide and 151mg Tris-base, add water to 8mL, with the HCl accent pH to 6.8 of 6mM, water is settled to 10mL; After mixing, be transferred in the centrifuge tube, add 10% ammonium persulphate that 50 μ L prepared the same day and the N of 5 μ L, N, N ', N '-Tetramethyl Ethylene Diamine, vibration mixes 20s, mixes back ultrasonic degas 10s, obtains concentrating sol solution, to concentrate sol solution and inject sheet glass, afterwards comb is inserted in the sheet glass, solidify, comb is extracted until gel, form sample holes, electrophoresis is finished with ionic liquid-polyacrylamide gel preparation.
Table 2 adds ion liquid quality
(2) pre-treatment protein example
With ten times of human serum dilute with waters, and with 2 * SDS-sample buffer (available from sky root biochemical technology company limited) volume ratio was mixed in 1: 1, after mixing, 95 ℃ of heating in water bath 5min cool off under the room temperature, obtain protein example.
(3) preparation protein electrophorese damping fluid and upward sample
Add Tris-base, 14.4g glycine and the 1g sodium lauryl sulphate of 3.03g in the 1L volumetric flask, the water constant volume prepares the protein electrophorese damping fluid; Assemble electrophoresis chamber then, the electrophoretic buffer of pouring into did not have the short slab of gum-making rack sheet glass, utilized microsyringe that the pretreated protein example of 10 μ L is injected sample holes respectively.
(4) electrophoresis
Connect electrophoresis apparatus.At first use the voltage electrophoresis of 100V, to the stall limit of tetrabromophenol sulfonphthalein arrival separation gel and concentrated glue, regulating voltage arrives the gel bottom to 140V until the tetrabromophenol sulfonphthalein electrophoresis then.
(5) dyeing and decolouring
Electrophoresis peels gel in water respectively after finishing from sheet glass, water cleans twice afterwards, dyes; Use 0.5% coomassie brilliant blue R250 staining fluid dyeing 1h during dyeing, use 20% methyl alcohol and 20% acetic acid mixed solution to decolour then, until removing the background background color.
In addition, carry out parallel control experiment SDS-PAGE.
Experimental result shown in Fig. 4,5 and 6, [the C that Fig. 4 prepares for embodiment 2
4Mpd] the Br mass concentration is 0.05% [C
4Mpd] Br-PAG, [C
4Mprd] the Br mass concentration is 0.05% [C
4Mprd] Br-PAG and [C
4Mim] the Br mass concentration is 0.05% [C
4Mim] the Br-PAG electrophorograms and its parallel control experiment SDS-PAGE that separate ten times of dilution human serums separate ten times of electrophorograms that dilute human serums.[the C that Fig. 5 prepares for embodiment 2
4Mpd] the Br mass concentration is 0.3% [C
4Mpd] Br-PAG, [C
4Mprd] the Br mass concentration is 0.3% [C
4Mprd] Br-PAG and [C
4Mim] the Br mass concentration is 0.3% [C
4Mim] the Br-PAG electrophorograms and its parallel control experiment SDS-PAGE that separate ten times of dilution human serums separate ten times of electrophorograms that dilute human serums.[the C that Fig. 6 prepares for embodiment 2
4Mpd] the Br mass concentration is 1.0% [C
4Mpd] Br-PAG, [C
4Mprd] the Br mass concentration is 1.0% [C
4Mprd] Br-PAG and [C
4Mim] the Br mass concentration is 1.0% [C
4Mim] the Br-PAG electrophorograms and its parallel control experiment SDS-PAGE that separate ten times of dilution human serums separate ten times of electrophorograms that dilute human serums.Electrophoresis result shows, compare with parallel control experiment SDS-PAGE, the ionic liquid-polyacrylamide gel of present embodiment preparation also utilizes it to carry out ionic liquid-sodium dodecyl sulfate-polyacrylamide gel electrophoresis the protein that is lower than 66.2KDa in the human serum is had tangible separation advantage.As can be seen from the figure, for the human serum protein of molecular weight ranges at 66.2～26.0KDa, the functional quality concentration range is imidazoles, pyridines and the pyrrolidines ionic liquid-polyacrylamide gel of alkyl carbon chain carbonatoms≤4 of 0.05～1.0% (w/v), and a resolution and band number average is better than its parallel control experiment SDS-PAGE.Wherein, described proteinic resolution and band number increase and increase along with ionic liquid concentration.With [C
4Mpd] Br-PAG is example, along with the increase of ionic liquid concentration, the clear protein band number in this zone increases to 6 by 2, when using 1.0%[C
4Mpd] during Br-PAG, isolating protein obtains best separation band number and resolution.For the human serum protein of molecular weight ranges at 26.0～12.0KDa, the mass concentration scope is that imidazoles, pyridines and the pyrrolidines ionic liquid-polyacrylamide gel of alkyl carbon chain carbonatoms≤4 of 0.3～1.0% (w/v) is more suitable, and a resolution and band number average is better than its parallel control experiment SDS-PAGE.Wherein, resolution and band number increase and increase along with ionic liquid concentration.As can be seen, the ionic liquid of lower concentration (0.05%) can not improve the proteinic resolution of this molecular weight ranges and separate the band number with increasing among Fig. 4; When Fig. 5 uses 0.3% ionic liquid-polyacrylamide gel, this regional protein band number increases to 2 by 1, when using 1.0% ionic liquid-polyacrylamide gel, this regional protein band number increases to 3 by 2, and this moment, institute's isolated protein obtained best separation band number and resolution.
The present invention includes but be not limited to above embodiment, every any being equal to of carrying out under the spirit and principles in the present invention, replace or local improvement, all will be considered as within protection scope of the present invention.
Claims (10)
1. an ionic liquid-polyacrylamide gel mainly comprises monomer, linking agent, gel buffer system, catalyzer and accelerator, it is characterized in that: also comprise ionic liquid; Wherein, described ionic liquid is the ionic liquid of alkyl carbon chain carbonatoms≤4 on the positively charged ion, and described ion liquid mass concentration is 0.05～1.0% (w/v).
2. a kind of ionic liquid-polyacrylamide gel according to claim 1 is characterized in that: on the described positively charged ion ionic liquid of alkyl carbon chain carbonatoms≤4 be alkyl carbon chain carbonatoms≤4 on the positively charged ion glyoxaline ion liquid, pyridines ionic liquid or pyrrolidines ionic liquid one of them.
3. a kind of ionic liquid-polyacrylamide gel according to claim 1 is characterized in that: described ion liquid mass concentration is used for the protein of isolated molecule weight range at 26.0～12.0KDa when being 0.3～1.0% (w/v).
4. a kind of ionic liquid-polyacrylamide gel according to claim 1 is characterized in that: described ionic liquid is the pyridines ionic liquid of alkyl carbon chain carbonatoms≤4 on the positively charged ion, and mass concentration is 1.0% (w/v).
5. preparation method as the described ionic liquid-polyacrylamide gel of claim 1～4, it is characterized in that: ionic liquid, monomer, linking agent and gel buffer system are mixed obtaining mixing solutions earlier, again catalyzer and accelerator being added described mixing solutions mixes, after solidifying, can obtain a kind of ionic liquid-polyacrylamide gel of the present invention.
6. purposes as the described ionic liquid-polyacrylamide gel of claim 1～4, it is characterized in that: described 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) prepares a kind of ionic liquid-polyacrylamide gel;
(2) SDS pre-treatment protein example;
(3) preparation protein electrophorese damping fluid and upward sample;
(4) electrophoresis;
(5) dyeing and decolouring;
Specifically, described electrophoresis method mainly comprises:
(1) preparation ionic liquid-polyacrylamide gel
Assembling sheet glass gum-making rack, earlier ionic liquid, monomer, linking agent and gel buffer system are mixed and obtain mixing solutions, again catalyzer and accelerator are added described mixing solutions after mixing 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 the sheet glass, solidify until gel, comb is extracted, form sample holes;
(2) pre-treatment protein example
Add sample buffered soln in protein example, mix post-heating, cooling obtains the pre-treatment protein example then;
(3) preparation protein electrophorese damping fluid and upward sample
The assembling electrophoresis apparatus, preparation protein electrophorese damping fluid also adds in the 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 sample on the protein example injector tunnel;
(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 finished is until removing the background background color.
7. the purposes of a kind of ionic liquid-polyacrylamide gel according to claim 6 is characterized in that: the both sides and the bottom that seal the gum-making rack sheet glass when assembling gum-making rack in the step (1) with Vaseline.
8. the purposes of a kind of ionic liquid-polyacrylamide gel according to claim 6 is characterized in that: add sample buffered soln in the step (2) in the protein example, after mixing, at 95 ℃ of heating in water bath 5min, cool off under the room temperature; The mass concentration of protein example is 0.1～0.5mg/mL.
9. the purposes of a kind of ionic liquid-polyacrylamide gel according to claim 6 is characterized in that: the last sample volume of protein example is 5～10 μ L in the step (3).
10. the purposes of a kind of ionic liquid-polyacrylamide gel according to claim 6 is characterized in that: after electrophoresis finishes in the step (5), in water gel is peeled from sheet glass.
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