CN117659115A - Method for removing hetero electrolyte by electric field force and application - Google Patents
Method for removing hetero electrolyte by electric field force and application Download PDFInfo
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- CN117659115A CN117659115A CN202311588573.6A CN202311588573A CN117659115A CN 117659115 A CN117659115 A CN 117659115A CN 202311588573 A CN202311588573 A CN 202311588573A CN 117659115 A CN117659115 A CN 117659115A
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Abstract
The invention provides a method for removing a hetero electrolyte by electric field force and application thereof. The method has the advantages that: 1. the method is rapid, and the mixed electrolyte can be removed instantaneously; 2. the purity of the separated substances is high; 3. the operation is simple; 4. the external conditions are easy to control, which is favorable for maintaining the activity of the biological macromolecular substances; 5. the separation and the enrichment of the target are integrated; 6. separating and enriching target substances in a large batch; 7. reducing the cost of separating and/or enriching the target. The application of the method is as follows: the method has wide application in experiments such as a Pulldown experiment, an Immunoprecipitation (IP), a Co-Immunoprecipitation (Co-IP), a chromatin Immunoprecipitation (Chromatin Immunoprecipitation, chIP), an RNA binding protein Immunoprecipitation (RNA Binding Protein Immunoprecipitation, RIP) and the like; also has wide application in separating and enriching protein, nucleic acid (DNA, RNA, etc.), sugar (monosaccharide, oligosaccharide, polysaccharide, etc.).
Description
[ field of technology ]
The invention belongs to the technical field of analytical chemistry and biology, and particularly provides separation methods for removing adsorbed polyelectrolyte by electric field force. Applications include, but are not limited to: protein, nucleic acid (DNA, RNA, etc.), sugar (monosaccharides, oligosaccharides, polysaccharides, etc.) separation and/or enrichment; it is also widely used in experiments such as Pulldown, immunoprecipitation (IP), co-Immunoprecipitation (Co-IP), chromatin Immunoprecipitation (Chromatin Immunoprecipitation, chIP), and RNA-binding protein Immunoprecipitation (RNA Binding Protein Immunoprecipitation, RIP).
[ background Art ]
With the development of genome sequencing technology, the research on the sequence, structure and function of biological genome is rapidly developed, and the acquisition of high-purity, high-content and high-integrity biological genome DNA is a primary premise that the genome sequencing technology is applied. With the development of biology, research on RNA, a transcription product of a gene, is becoming more and more popular with researchers.
More and more DNA, RNA, and co-extraction methods of both have been developed. Specifically, the traditional extraction methods of biological genome DNA mainly comprise CTAB and SDS methods; common biological RNA extraction methods mainly comprise Trizol method, guanidine thiocyanate/phenol method, SDS/phenol method, guanidine hydrochloride method and the like.
The conventional DNA and/or RNA extraction methods have drawbacks: complicated operation, time consumption, low purity, and unfavorable mass separation and enrichment of DNA and/or RNA. In recent years, various commercialized microspheres and magnetic nanomaterials have been developed for separating and/or enriching DNA and/or RNA, which can overcome the disadvantages of the conventional methods for separating and/or enriching DNA and/or RNA. The extraction of DNA and/or RNA has greatly advanced. However, the process itself also has disadvantages: the microsphere and the magnetic nano material for separating and/or enriching DNA and/or RNA can also adsorb proteins and/or saccharides and the like, and especially the nano material has large specific surface area and strong adsorption capacity and is easier to adsorb impurities.
In recent years, with the completion of the human genome project, efforts to analyze huge human genome information have been proposed on the schedule. Proteins have tremendous biological information as gene transcription and translation products, and research of proteomics is carried out to obtain information of protein expression level, so that the proteins are favored and paid attention to by researchers. As the premise and the basis of proteomics research, the separation and purification of the protein has very important biological significance. Currently, researchers have developed a series of methods for protein isolation and/or enrichment using differences in the nature of the protein molecules themselves: the different sizes of protein molecules establish methods such as ultrafiltration, gel filtration, colloid electrophoresis and the like; the different charges of protein molecules develop methods such as ion exchange chromatography, gel electrophoresis, isoelectric precipitation and the like; affinity chromatography methods and the like are developed due to the difference of the hydrophilicity and hydrophobicity of protein molecules.
However, these methods have certain disadvantages when used for protein separation and/or enrichment, such as gel filtration, which generally requires preparation and handling in advance, and is time-consuming and complicated to operate; the packing and pretreatment of the ion exchange column of the ion exchange chromatography are also complicated; the separation of proteins by affinity chromatography is not limited to specific adsorption.
Pure microspheres (Beads) or nano-material-antibody or protein molecular probe complexes are required to be prepared in experiments such as a Pulldowr experiment, an IP experiment, a Co-IP experiment, a ChIP experiment, a RIP (RIP) and the like, and after the molecular probes are combined with a target object, the non-specific adsorption proteins can be removed by the method provided by the invention to retain the specifically combined target proteins. However, experiments have been left to date due to the presence of non-specific adsorption of chemical groups or very large specific surface areas on the surface of microspheres or nanomaterials.
The invention aims to solve the technical problem of eliminating nonspecific adsorption on the surfaces of microspheres or nano materials, and provides a few cheap, rapid and simple methods for eliminating nonspecific adsorption of a hetero-electrolyte based on the principle that charged ions are subjected to electric field force in an electric field and application thereof.
[ invention ]
The present invention aims to overcome the above-mentioned drawbacks of the prior art and provide methods for removing nonspecifically adsorbed polyelectrolytes by electric field forces and applications thereof: has wide application in separating and/or enriching protein, nucleic acid (DNA, RNA, etc.), sugar (monosaccharide, oligosaccharide, polysaccharide, etc.); the method has wide application in experiments such as PullDown experiments, IP experiments, co-IP experiments, chIP experiments, RIP and the like.
1. A method for the separation and/or enrichment of a target and/or for the preparation of a matrix containing the target and the matrix, according to claim 1, characterized in that it comprises the following steps:
a) Incubating the substrate in a solution of the mixture comprising the target to bind the target to the substrate;
b) Removing the hetero electrolyte under the action of one or more than one electric field force, and retaining the matrix and the target;
c) And/or eluting the target from the substrate to achieve the purpose of separating or purifying the target;
d) And/or the matrix bound to the target as a whole for further experiments.
For the purposes of the present invention, the term "hetero-electrolyte" means a compound that is capable of dissociating charged ions in aqueous solution rather than isolated and/or enriched targets.
For the purposes of the present invention, the term "target" means a substance that needs to be isolated and/or enriched, such as: nucleic acids (DNA, RNA, etc.), proteins, carbohydrates, etc.
For the purposes of the present invention, the term "nucleic acid" means a nucleic acid comprising, but not limited to: naturally, preferably isolated, linear, branched or circular nucleic acids such as RNA, in particular mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA, microRNA or ribozymes, DNA, plasmid DNA, mitochondrial DNA and the like.
For the purposes of the present invention, the term "matrix" means a solid substance capable of specifically binding to a target, including but not limited to: various commercial DNA, RNA, protein-enriched microspheres, magnetic microspheres, nanomaterials, and the like.
As a preferred option, the target and substrate are washed with a washing liquid to remove some of the non-specifically adsorbed polyelectrolyte that is soluble in the washing liquid prior to removal of the polyelectrolyte by electric field forces.
The substrate of claim 2, including but not limited to: various commercially available microspheres, such as: various microspheres for isolating and/or enriching DNA, RNA and proteins, various commercial magnetic microspheres, such as: various magnetic microspheres for isolating and/or enriching DNA, RNA and proteins, various commercial magnetic nanomaterials, such as: various nano-materials for separating and/or enriching DNA, RNA and protein, etc. Because the surface of the microsphere or the magnetic nano material is modified by some chemical groups, non-specific adsorption can occur under the actions of static electricity, hydrophobic action and/or van der Waals force, and the like, particularly the nano material has very large specific surface area, and the non-specific adsorption is more serious.
A method as claimed in claim 3, wherein: labels contained by the target include, but are not limited to: the sugar chain of glycoprotein or glycopeptide, the phosphate group of phosphorylated protein or phosphorylated peptide, the ubiquitin group of ubiquitin protein, the hydroxyl group on sugar, nucleic acid including DNA, RNA, etc., HIS Tag recombinant protein (fusion Tag composed of 6 histidine residues), flag-Tag recombinant protein (fusion Tag composed of 8 amino acid hydrophilic polypeptide (DYKDDDK)), aviTag recombinant protein (fusion Tag composed of 15 amino acid short peptide), SNAP-Tag recombinant protein (obtained from human O6-methylguanine-DNA methyltransgerase), GST recombinant protein (glutathione-sulfhydryltransferase) Tag, C-Myc Tag recombinant protein (small Tag containing 11 amino acids), etc. The target is bound to the substrate via the tag, forming a new complex.
The method as set forth in claim 4, wherein: the polyelectrolytes include, but are not limited to: proteins, peptides, nucleic acids, inorganic salts, and the like.
As described in claim 5 and 6, the method is simple and has few components of the hetero-electrolyte, and the electric field force is applied for 1 time to remove the hetero. Wherein, when the hetero-electrolyte is an amphoteric substance, the pH value of the electrophoresis liquid and the isoelectric Point (PI) of the amphoteric substance in the hetero-electrolyte cannot be equal. However, for the hetero-electrolyte components to be more and/or contain amphoteric substances, electric field force is required for removing impurities 2 times and/or more, and the PH of the electrophoresis liquid 1 and the PH of the electrophoresis liquid 2 and/or the PH of other electrophoresis liquids cannot be equal. When the PH value of the electrophoresis liquid is equal to the PI of the amphoteric substance, the amphoteric substance is uncharged and is not acted by the electric field force in the electric field, so that the separation of the polyelectrolyte and the target substance can not be realized; when there are multiple ampholytic substances, many different PIs will appear, and when the PH of the electrophoretic fluid is close to PI, the electric field force to which the hetero electrolyte is subjected is also small. If the electric field force of the polyelectrolyte is smaller than the adsorption force of the polyelectrolyte on the surface of the substrate, the purpose of separating the polyelectrolyte from the target object cannot be achieved. Therefore, on the premise of keeping the biological activity of the target object, the larger the PH value difference of the 2 kinds of electrophoresis solutions is, the better the nonspecific adsorption is, and the more thorough the removal is.
According to a preferred embodiment, the number of times the electric field force removes the polyelectrolyte is one or more times, and for a system of simple composition, a preferred number of times the electric field force removes the polyelectrolyte is 1 time; the more preferable number of times of removing the hetero electrolyte by the electric field force for the system having a complicated composition and containing a plurality of amphoteric substances is 2 times.
As described in claim 7, when the binding force of the polyelectrolyte to the substrate is large, and/or the concentration of the surfactant or organic matter in the liquid is increased, the binding force of the polyelectrolyte to the substrate is reduced, which is advantageous for removing the polyelectrolyte. In addition, the concentration of salt in the liquid is/are improved, the binding force between impurities and a matrix is reduced, and the removal of the polyelectrolyte is facilitated.
The method of claim 8, wherein the electric field is a uniform electric field or a non-uniform electric field.
The method according to claim 9, wherein the medium for transmitting the electric field force is electrophoretic fluid or vacuum or thin air.
As set forth in claim 10, under the action of an electric field force,
-and/or the matrix (diameter D1) bound to the target remains outside the gel (diameter D2), the hetero-electrolyte (diameter D3) entering the gel, wherein D1 > D2 > D3;
-and/or the solid substance (diameter D1) bound to the target remains in the pocket containing a plurality of small holes (pore diameter D4), the polyelectrolyte (diameter D3) moving towards the two poles, wherein D1 > D4 > D3;
And/or the substrate to which the target is bound is attracted to a magnet or a device generating magnetic force, such as an electromagnet or the like, the hetero-electrolyte being moved towards the two poles. One or more of the cases of claims 8, 9, 10 taken together form a separation and/or enrichment method. Such as: the medium of the electrostatic field, which is transmitted to the force of the electric field, is liquid and the solid matter (diameter D1) containing the target object is reserved in a pocket containing a plurality of small holes (diameter D4), and the hetero electrolyte (diameter D3) moves to two poles, wherein D1 is more than D4 and more than D3, and the solid matter and the hetero electrolyte can be combined into a novel separation and/or enrichment method.
The method as set forth in claim 11, wherein: voltage: 50V-30000V; current flow: 0mA and/or 10 mA-10A. When large-scale processing of samples is performed, high voltages are required to provide sufficient electric field force to remove the parasitic electrolyte. Under even strong electric field, the charged hetero-electrolyte is moved to two poles by vacuum or thin air-electrophoresis liquid-vacuum or thin air transmission electric field force, and the target and the substrate stay in the small bag with micropores. The essence of the method is that the charges of the hybrid electrolyte are redistributed to the two poles of the uniform electric field, and no current is formed, so that the current is 0mA.
The method as set forth in claim 12, wherein: and/or cleaning the substrate and the target to further remove the parasitic electrolyte adsorbed thereon. Because the matrix and the target can nonspecifically adsorb some polyelectrolyte, the adsorbed polyelectrolyte can be removed by a cleaning solution for facilitating subsequent treatment.
The method as set forth in claim 13, wherein: when the target is an active protein, the separation conditions are as follows:
the liquid cannot contain more than the concentration of denaturing agents that reduce the activity of the biological macromolecules, such as: the liquid does not contain Sodium Dodecyl Sulfate (SDS), or the content thereof is extremely low;
-and/or the liquid contains protein inhibitors such as: phenylmethylsulfonyl fluoride (phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluorid, PMSF), pepstatin, leupeptin (leupeptin), trypsin inhibitor (aprotinin), mixed protease inhibitor, EDTA, disodium edetate, and the like;
-and/or the liquid contains a protein protecting agent, such as: the monohydric alcohol is methanol or ethanol, the dihydric alcohol is ethylene glycol, propylene glycol and butanediol, the trihydric alcohol is glycerol, the glycol polymer is polyethylene glycol, polypropylene glycol and a copolymer of ethylene glycol and propylene glycol, the polyhydroxy monosaccharide is glucose, fructose, mannose and xylose, the disaccharide is sucrose, lactose and maltose, and the polysaccharide is starch, glucan and the like. Preferred is glycerol;
-and/or the temperature is controlled between 0 ℃ and 25 ℃, most preferably at 4 ℃;
and/or absorbing the polyelectrolyte by agarose gel or non-denaturing PAGE or the like.
The method as set forth in claim 14, wherein: the separation method can be applied in the following experiments including but not limited to: pulldown experiments, immunoprecipitation experiments (IP), co-Immunoprecipitation experiments (Co-Immunoprecipitation, co-IP), chromatin Immunoprecipitation (Chromatin Immunoprecipitation, chIP), RNA-binding protein Immunoprecipitation (RNA Binding Protein Immunoprecipitation, RIP), and the like.
In the Pulldown experiment, it is necessary to prepare a complex of pure microspheres or nanomaterials with a molecularly tagged protein a in advance without allowing the adsorption of the hybrid protein on the complex. Specifically, the tag added genes for connecting the microspheres or the nanometer and the protein A are connected into the genome of an expression system (four large protein expression systems: an escherichia coli expression system, a yeast expression system, an insect cell expression system and a mammal expression system) through genetic molecular recombination engineering, recombinant proteins are expressed, cells are cracked nondenaturing or collecting the recombinant proteins expressed in the supernatant, functional microspheres or nanometer materials are added into a mixture of the cracked cells or the supernatant containing the recombinant proteins, and the mixture of the microspheres or the nanometer materials and the protein A is formed through incubation. The method for removing the hetero-electrolyte provided by the invention is used for removing the hetero-protein, so that pure microsphere or nano material and protein A complex is prepared. If the foreign proteins are not removed cleanly, the experiment can be stopped before or the subsequent analysis and/or experiment can be greatly reduced. Then, the complex is added to a protein extraction mixture to be analyzed for incubation, so that protein B, and/or protein C, and/or protein d, which interact with protein a, are combined to form a new complex, and the adsorbed foreign proteins are removed again by the impurity removal method provided by the present invention. This greatly reduces subsequent analysis and verification effort.
In the IP experiment, after the antibody is combined with the corresponding protein in the cell lysate or the expression supernatant, the antibody is incubated with protein A/G (ProteinA/G) or a secondary antibody coupled microsphere or nanomaterial, the microsphere or nanomaterial-protein A/G or a secondary antibody-target protein complex is obtained through centrifugation, and/or the absorbed hybrid protein is removed by the method for removing the hybrid electrolyte provided by the invention after the sediment is washed. This greatly reduces subsequent analysis and verification effort.
In Co-IP experiments, many protein-protein interactions present within intact cells are retained when the cells are lysed under non-denaturing conditions. When protein a is immunoprecipitated with an antibody to protein a previously immobilized on a functional microsphere or nanomaterial, protein B that binds to protein a in vivo can also precipitate together. And then protein denaturation and separation are carried out to detect the protein B, so as to prove the interaction between the protein B and the protein B. Some of the hetero-proteins are adsorbed on the new microsphere or nanomaterial-protein A antibody-protein A-protein B complex, and the adsorbed hetero-proteins are removed by the method for removing the hetero-electrolyte provided by the invention. This greatly reduces subsequent analysis and verification effort.
In ChIP experiments, the basic principle is to fix a protein-DNA complex in a living cell state, randomly cut the complex into small chromatin fragments within a certain length range, precipitate the complex by an immunological method, specifically enrich a DNA fragment bound to a target protein, and obtain information of protein-DNA interaction by purifying and detecting the target fragment. Some of the hetero-proteins are adsorbed on the new microsphere or nanomaterial-protein A/G or secondary anti-antibody-protein-DNA complex, and the adsorbed hetero-proteins are removed by the method for removing the hetero-electrolyte provided by the invention. This greatly reduces subsequent analysis and verification effort.
In RIP experiments, the basic principle is that a protein-RNA complex in cells is precipitated by an immunological method, RNA fragments bound with target proteins are specifically enriched, and information of interaction between the proteins and the RNA is obtained by purifying and detecting the target fragments. Some of the hetero-proteins are adsorbed on the new microsphere or nanomaterial-protein A/G or secondary anti-antibody-protein-RNA complex, and the adsorbed hetero-proteins are removed by the method for removing hetero-electrolytes provided by the invention. This greatly reduces subsequent analysis and verification effort.
The label-containing target of claim 15, including but not limited to: glycoprotein or glycopeptide, phosphorylated protein or phosphorylated peptide, nucleic acid (DNA, RNA, etc.), sugar (monosaccharide, oligosaccharide, polysaccharide, etc.), ubiquitin or ubiquitinated protein, protein with antibody, HIS Tag recombinant protein (fusion Tag composed of 6 histidine residues), flag-Tag recombinant protein (fusion Tag composed of 8 amino acid hydrophilic polypeptide (dykdddk)), aviTag recombinant protein (fusion Tag composed of 15 amino acid short peptide), SNAP-Tag recombinant protein (obtained from human O6-methylguanine-DNA methyltransgerase), GST recombinant protein (glutathione sulfhydryl transferase) Tag, C-Myc Tag recombinant protein (small Tag containing 11 amino acids), and the like.
For example, the invention is useful in the isolation or purification of glycoproteins or glycopeptides from proteins. Methods of separation and/or enrichment of glycopeptides or glycoproteins related to the present invention include, but are not limited to: lectin affinity chromatography, hydrazide method, boric acid method, and titanium dioxide (TiO 2 ) Enrichment, hydrophilic interaction liquid chromatography (HILIC), etc.
Lectin affinity chromatography generally immobilizes lectins on a support such as agarose, silica or polyhydroxy polymer, and lectins specifically recognize sugar chains on a substrate glycoprotein or glycopeptide and bind thereto, and remove adsorbed non-glycoproteins and/or non-glycopeptides through a chromatographic column.
In the hydrazide method, the cis-dihydroxy of the glycan is oxidized by periodate and then covalently coupled to the resin to which the hydrazide group is fixed. In contrast to lectin affinity methods, hydrazide capture is non-specific and can enrich all glycoconjugates.
Boric acid processes, boric acid is capable of covalent but reversible chemical reaction with saccharides containing 1-2 and 1-3 cis-dihydroxy groups (e.g., mannose, glucose, and galactose) to form stable cyclic esters. Boric acid recognizes glycans as nonspecific, i.e., as well as glycans with various branches, as well as linear glycans, and as well as monosaccharides, thereby enabling unbiased enrichment of various N-and O-linked glycopeptides.
Microspheres, magnetic microspheres, nanomaterials capable of separating and/or enriching glycoproteins or glycopeptides may also be used to separate and/or enrich carbohydrates. The method provided by the invention can also be used for removing nonspecifically adsorbed hybrid proteins or nucleic acids and the like.
Titanium dioxide (TiO) 2 ) Enrichment method, tiO 2 Are used for glycopeptide enrichment due to their affinity for sialic acid. Since both phosphopeptide and glycopeptide are bound to TiO 2 The removal of phosphate modifications by phosphatase (NEB provides Lambda protein phosphatase for protein dephosphorylation) pretreatment is thus beneficial for improving the efficiency of glycopeptide enrichment.
Hydrophilic liquid chromatography (HILIC) is a promising separation and enrichment method for separating glycoproteins and glycopeptides and glycans derived from glycoproteins. HILIC is characterized by the use of a hydrophilic stationary phase based on cation exchange, anion exchange, zwitterionic interactions and agarose gels, and a relatively hydrophobic organic mobile phase. Due to the influence of the carbohydrate moiety having a hydrophilic nature in the structure, the glycopeptides can be more firmly bound to the HILIC column and thus separated from the non-glycosylated peptides in the proteolytic digestion products. The hydrophilicity of the oligosaccharides in the glycopeptides makes them excellent targets for HILIC isolation.
The various glycoprotein or glycopeptide isolation and/or enrichment methods described above share the common feature: the glycoprotein or glycopeptide can bind to the functionalized substrate, and there is also non-specific adsorption. The method provided by the invention can be used for removing nonspecifically adsorbed hybrid proteins.
More specifically, the isolation and/or enrichment methods of phosphorylated proteins or phosphorylated peptides relevant to the present invention include, but are not limited to: solid phase metal ion affinity chromatography (immobilized metal affinity chromatograph, IMAC) and metal oxide affinity chromatography (metal oxide affinity chromatography, MOAC).
IMAC: by means of transition metal cations, e.g. Fe 3+ 、Ga 3+ 、Zr 4+ And the like, as an affinity reagent, to bind to anions. Ti (Ti) 4+ Ions are an emerging application in this class of agents. These metal cations are immobilized by chelation to magnetic beads or silicon particles, which allow the phosphorylated peptide to remain during removal of the non-phosphorylated peptide.
MOAC: enrichment of phosphorylated peptide fragments is performed by utilizing the property that oxidized metal can bind to oxygen in phosphate groups. Oxides of titanium (TiO, ti 2 O 3 、TiO 2 ) Is the most commonly used MOAC reagent, and furthermore Fe 3 O 4 It is also common.
The separation and/or enrichment method of 2 phosphorylated proteins or phosphorylated peptides as described above has a common feature: non-specific adsorption of either phosphorylated proteins or phosphorylated peptides can be bound to the functionalized substrate. The method provided by the invention can be used for removing nonspecifically adsorbed hybrid proteins.
More specifically, nucleic acid isolation and/or enrichment methods related to the present invention include, but are not limited to: functionalized microspheres, magnetic microspheres and nanomaterials can specifically recognize and efficiently bind nucleic acids (DNA, RNA, etc.), thereby isolating and/or enriching nucleic acids. But also nonspecific adsorption, especially adsorption of proteins, occurs. The method provided by the invention can be used for removing nonspecifically adsorbed proteins.
More specifically, methods of isolation and/or enrichment of antibody proteins present in connection with the present invention include, but are not limited to: the functionalized microspheres, magnetic microspheres and nanomaterials bind to antibodies and then the protein extraction mixture containing the substrate protein is incubated to form new functionalized microspheres or nanomaterial-protein a/G or secondary anti-antibody-protein complexes. The method provided by the invention can be used for removing nonspecifically adsorbed hybrid proteins. Among them, traditional methods of ubiquitin protein isolation and/or enrichment are also based on the principle of antibody and antigen specific recognition immune responses.
More specifically, a specific tag is introduced on the gene of the target protein by DNA molecular recombinant genetic engineering for isolating and/or enriching the target protein. The tag incorporated includes, but is not limited to: HIS tagged recombinant protein (fusion Tag composed of 6 histidine residues, flag-Tag recombinant protein (fusion Tag composed of 8 amino acid hydrophilic polypeptide (DYKDDDDK)), aviTag recombinant protein (fusion Tag composed of 15 amino acid short peptide), SNAP-Tag recombinant protein (obtained from human O6-methylguanine-DNA methyltransgerase), GST recombinant protein (glutathione sulfhydryl transferase) Tag, C-Myc tagged recombinant protein (small Tag containing 11 amino acids). In practical application, HIS Tag and GST Tag are most commonly used.
The method as set forth in claim 16, wherein the electrophoresis liquid used is characterized in that:
-at least contain H 2 O；
-and/or contain acids, including but not limited to: hydrochloric acid, glacial acetic acid, sulfuric acid, etc., are preferred, and hydrochloric acid is mainly used for adjusting the pH of the electrophoretic fluid.
-and/or contain a base, including but not limited to: sodium hydroxide, tris-base, etc., preferably Tris-base, for stabilizing the pH of the electrophoretic fluid.
-and/or containing salts or salt mixtures, including but not limited to: sodium chloride, sodium acetate, potassium chloride, and the like, preferably sodium chloride, provides a polar environment that facilitates removal of the polyelectrolyte.
-and/or one or more water-soluble organics, preferably including but not limited to: glycerol, glycine, imidazole, and the like, with glycerol being more preferred. Mainly, when active protein is separated and/or prepared, the glycerol can play a role in protecting the activity of the protein.
And/or one or more surfactants, namely cationic, anionic, zwitterionic and/or nonionic. Based on the total volume of the reagent, the dosage of the reagent is as follows: the weight/volume is between 0 and 50%, preferably polyvinylpyrrolidone 40 (PVP 40), 4-nonylphenyl-polyethylene glycol 40 (NP-40), triton X-100 (Triton X-100), etc., and the dosage thereof is as follows: the weight/volume is between 1 and 10 percent. Particularly, for removing the polyelectrolyte with strong hydrophobicity, the adsorption force of the matrix compound on the polyelectrolyte can be reduced by increasing the content of the surfactant, and the removal of the polyelectrolyte is facilitated.
And/or one or more buffer compounds, the pH of which may be between 3 and 11, preferably chosen from, but not limited to: TRIS (hydroxymethyl) aminomethane (TRIS), N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), N-bis (2-hydroxyethyl) glycine (BICINE), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2- (N-morpholino) ethanesulfonic acid (MES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffers, sodium acetate buffers, methyl acetate buffers, and the like, preferably TRIS, phosphate buffers, sodium acetate buffers, methyl acetate buffers, and the like, at a concentration of: 1 mM-10 mM. The method mainly provides an electrophoresis environment with stable pH value, is favorable for removing the hetero electrolyte, and particularly causes the pH value of the electrophoresis liquid to change due to electrolytic reaction of the electrophoresis liquid when current passes through the electrophoresis liquid. This disadvantage can be overcome by the addition of surfactants.
-and/or one or more chelating compounds, preferably selected from the group comprising, but not limited to: N-acetyl-L-cysteine, ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), ethylenediamine-N, N ' -disuccinic acid (EDDS), 1, 2-bis (o-aminophenoxy) ethane-N, N, N ', N ' -tetraacetic acid (BAPTA), and phosphonate chelating agents (including, for example, but not limited to nitrilotris (methylene) phosphonic acid (NTMP), ethylenediamine tetra (methylene phosphonic acid) (EDTMP), diethylenetriamine penta (methylene) phosphonic acid (DTPMP), 1-hydroxyethylidene-1, 1-diphosphine (HEDP), and the like). Preferred are EDTA, DTPA, at concentrations of: 5 mM-100 mM. When the target is DNA, the addition of chelating compounds, especially EDTA, may be necessary to stabilize the DNA.
-and/or one and/or more RNase a inhibitor mixture, such as: including but not limited to some of the reducing agents disclosed in U.S. patent No. 6825340 or U.S. patent No. 677720. In some embodiments, the commonly used RNase a inhibitor is diethyl pyrocarbonate (DEPC). Preferably 2-mercaptoethanol, 8-hydroxyquinoline, DEPC, etc. When the target is RNA, the addition of RNase A inhibitor cocktail can prevent or slow degradation of RNA.
The method as set forth in claim 17, wherein: one and/or more of the cases taken from claims 8,9 and 10 can be combined to form a new method for removing the polyelectrolyte. Such as: the uniform electric field is selected from claim 8, the electrophoresis liquid and the vacuum or thin air are selected from claim 9, and the pocket with micropores is selected from claim 10, so that a new separation method can be combined. After the power is turned on, the hetero electrolyte moves to the two poles of the uniform electric field, and the target and the matrix stay in the pocket to separate the target or prepare the pure molecular probe.
The method as set forth in claim 18, wherein: glues include, but are not limited to: agarose gel, denaturing polyacrylamide gel (SDS-PAGE), non-denaturing polyacrylamide gel (PAGE), and the like. Gel action: 1. allowing the matrix bound to the target to stay outside the gel. 2. Absorbing hetero-electrolytes such as hetero-proteins, etc.
The cleaning solution of claim 19, wherein the composition of the cleaning solution is:
-at least contain H 2 O。
-and/or contain acids, including but not limited to: hydrochloric acid, glacial acetic acid, sulfuric acid, etc., preferably hydrochloric acid, to adjust the PH of the cleaning solution.
-and/or contain a base, including but not limited to: sodium hydroxide, tris-base, etc., preferably Tris-base, adjust the pH of the washing solution.
-and/or containing salts or salt mixtures, including but not limited to: sodium chloride, sodium acetate, potassium chloride and the like, preferably sodium chloride, increases the polar environment of the cleaning liquid and is beneficial to removing the polar hetero electrolyte.
And/or one or more water-soluble organic substances, preferably selected from the group comprising, but not limited to: glycerol, glycine, imidazole, etc., preferably glycerol, facilitate maintenance of protein activity.
And/or one or more surfactants, namely cationic, anionic, zwitterionic and/or nonionic. Based on the total volume of the reagent, the dosage of the reagent is as follows: weight/volume between 0 and 50%, preferably PVP40, NP-4), triton X-100, etc., in an amount of: the weight/volume is between 1 and 10 percent. Is favorable for removing the hetero electrolyte with strong hydrophobicity.
And/or one or more buffer compounds, the pH of which may be between 3 and 11, preferably chosen from, but not limited to: TRIS, TRICINF, BICINE, HEPES, PIPES, CHES, MES, MOPS and/or phosphate buffer, sodium acetate buffer, methyl acetate buffer, etc., preferably TRIS, phosphate buffer, sodium acetate buffer, methyl acetate buffer, etc., at a concentration of: 1 mM-10 mM. Is beneficial to maintaining constant PH environment of the target and the matrix.
-and/or one or more chelating compounds, preferably selected from the group comprising, but not limited to: N-acetyl-L-cysteine, EDTA, DTPA, EDDS, BAPTA, and phosphonate chelators (including, but not limited to NTMP, EDTMP, DTPMP, HEDP, etc., for example). Preferred are EDTA, DTPA, at concentrations of: 5 mM-100 mM. Preventing or slowing down DNA degradation.
-and/or one and/or more RNase a inhibitor mixture, such as: including but not limited to some of the reducing agents disclosed in U.S. patent No. 6825340 or U.S. patent No. 677720. In some embodiments, the commonly used RNase a inhibitor is DEPC. 2-mercaptoethanol, 8-hydroxyquinoline, DEP, and the like are preferable. Preventing or slowing down RNA degradation.
The isolated active protein of claim 20, comprising: acidic proteins, neutral proteins, and basic proteins. The electric field force applied to the electrophoresis liquid at a specific pH is different depending on the pH of the protein. However, the electric field force is negligible compared with the covalent bonding force of the target and the matrix.
The agarose gel of claim 21, more specifically 0.5% to 2%, more preferably 1% agarose; more specifically, 8-15% SDS-PAGE, and even more preferably 10-12% SDS-PAGE; more specifically, 8 to 15% of non-denaturing SDS-PAGE, more preferably 10 to 12% of non-denaturing SDS-PAGE, and the like. The proper gel concentration can absorb the hetero-electrolyte and can stay the matrix combined with the target outside the gel.
The method as set forth in claim 22, wherein: the target and matrix complex can be processed in large volumes to remove adsorbed polyelectrolytes.
The specific implementation steps are as follows:
a) Incubating the substrate in a solution of the mixture comprising the target to bind the target to the substrate;
b) Removing the hetero electrolyte under the action of one or more than one electric field force, and retaining the matrix and the target;
c) And/or eluting the target from the substrate to achieve the purpose of separating or purifying the target;
d) And/or the matrix containing the target as a whole for further experiments
The method for removing the hetero electrolyte by using the electric field force and the application thereof provided by the invention have at least the following beneficial effects:
1. the operation is simple, the time is saved, and the parasitic electrolyte can be removed instantaneously;
2. the purity of the separated and/or enriched target is high;
3. the external conditions are easy to control, which is favorable for maintaining the activity of biological macromolecules;
4. integrating the separation and/or enrichment process of the target object;
5. separating and enriching target substances in a large batch;
6. reducing the cost of the separated and/or enriched target;
7. the method can be used for separating and/or enriching proteins, nucleic acids (DNA, RNA, etc.), saccharides (monosaccharides, oligosaccharides, polysaccharides, etc.), etc.;
8. the method can be widely applied to Pulldown experiments, IP experiments, co-IP experiments, chIP experiments, RIP experiments and the like.
[ description of the drawings ]
FIG. 1 is a diagram showing gel electrophoresis of DNA and RNase A after electric field force impurity removal;
FIG. 2 is a diagram showing gel electrophoresis of DNA and RNase A after electric field force impurity removal;
FIG. 3 is a graph of gel electrophoresis comparing before and after fibronectin separation after electric field force removal;
[ detailed description ] of the invention
The invention is further described below with reference to the drawings and specific embodiments, which are only used to aid in the understanding of the invention.
Example 1-objects and substrates remain outside the gel, impurities enter the gel, liquid conductive electric field force impurity removal method
As described above, 200ul Boric Acid Beads 4FF (particle size: 45um to 165um of Changzhou Tiandi and Biotech Co., ltd.) was added to 200ul of a solution containing 50mM NH 4 HCO 3 In 225ng/uL DNA and 500ng/uL RNase A (non-glycoprotein) solution, incubating for 2 hours at room temperature, centrifuging at 3000rpm for 1 minute, discarding the supernatant, adding the precipitate into the middle square space of 10% SDS-PAGE gel (prepared SDS-PAGE gel, removing part of gel at the middle position by a craft knife, leaving a square space, of course other shapes, and the like), 80V, and after electrophoresis for 2 minutes, taking 2 parts of 5uL of Boric Acid beans 4FF after electric field force impurity removal, boiling for 10 minutes, and running 1% agarose gel and 10% SDS-PAGE gel respectively.
As can be seen from the results of FIG. 1, DNA was retained on the Beads without any residue of RNase A. Description: the electric field force impurity removal method provided by the invention can thoroughly remove the nonspecifically adsorbed RNase A.
Example 2-method for removing impurities by conduction of electric field force from liquid with objects and substrates left in the pocket and impurities left outside the pocket
As described above, 20ul Boric Acid Beads 4FF (particle size: 45um to 165 um) was added to 200ul of a solution containing 50mM NH 4 HCO 3 22.5ng/ul DNA and 500ng/ul RNase A (non-glycoprotein) solution, incubating for 2 hours at room temperature, centrifuging at 3000rpm for 1 minute, discarding supernatant, adding the precipitate into a pocket with a 3um nylon filter (Shanghai Xingjia purification Material Co.), and placing the pocket in a solution containing 50mM NH 4 HCO 3 In the solution, 100V electrophoresis was performed for about 5 seconds, 2 parts of the electric field-driven Boric Acid Beads 4FF were respectively taken and boiled in boiling water for 10 minutes, and 1% agarose gel and 10% SDS-PAGE gel were respectively run.
As can be seen from the results of FIG. 2, DNA was retained on the Beads without any residue of RNase A. Description: the electric field force impurity removal method provided by the invention can thoroughly remove the nonspecifically adsorbed RNase A.
Example 3-method for removing impurities by conduction of electric field force from liquid with objects and substrates left in the pocket and impurities left outside the pocket
As described above, 20ul Ni NTABeads 6FF (particle size: 45um to 165um of Changzhou Tiandi and Biotech Co., ltd.) was added to 200ul of a solution containing 50mM NH 4 HCO 3 In 831ng/uL of the fibronectin gene-transformed E.coli protein extract, incubation was performed at 4℃for 2 hours, centrifugation was performed at 3000rpm for 1 minute at 4℃and the supernatant was discarded, the precipitate was put into a pocket of a nylon filter membrane (Shanghai Xingjia purification material factory) having a pore size of 3um, and the pocket was placed in a pH 8.0 electrophoresis solution, and the impurities were removed by 400V electrophoresis for about 5 seconds. The bag was then placed in a pH 9.0 electrophoresis solution, and 400V was electrophoresed to remove impurities for about 5 seconds. Taking 1 part of Ni NTA bearings 6FF which is subjected to electric field force impurity removal, boiling for 10 minutes in boiling water, and running 10% SDS-PAGE gel.
As can be seen from the results of FIG. 3, fibronectin was retained on the Beads and no other hybrid proteins remained. Description: the electric field force impurity removing method provided by the invention can remove nonspecifically adsorbed proteins.
The results show that the electric field force impurity removing method provided by the invention has the advantages of good impurity removing effect, simplicity in operation, time saving, easiness in controlling external conditions, convenience in maintaining the bioactivity of biological macromolecules and the like, and has wide application.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, combinations, substitutions and alterations can be made to these embodiments without departing from the principles and structure of the present invention, the protection of which is defined by the appended claims and their equivalents.
Claims (22)
1. A method for separating and/or enriching a target substance by rapidly removing a hetero-electrolyte under the action of an electric field force and/or a method for preparing a substrate containing the target substance and the substrate, characterized by comprising the steps of:
a) Incubating the substrate in a solution of the mixture comprising the target to bind the target to the substrate;
b) Removing the hetero electrolyte under the action of one or more than one electric field force, and retaining the matrix and the target;
c) And/or eluting the target from the substrate to achieve the purpose of separating or purifying the target;
d) And/or the matrix bound to the target as a whole for further experiments.
2. The method according to claim 1 a), wherein: the matrix includes, but is not limited to: microspheres that bind to a target, such as: various commercial microsphere products, magnetic microspheres bound to targets, such as: various commercial magnetic microsphere products, magnetic nanomaterials associated with targets, such as: various commercialized magnetic nanomaterials, and the like.
3. The method according to claim 1 a), wherein: labels contained by the target include, but are not limited to: the sugar chain of glycoprotein or glycopeptide, the phosphate group of phosphorylated protein or phosphorylated peptide, the ubiquitin group of ubiquitin protein, the hydroxyl group on sugar, nucleic acid including DNA, RNA, etc., HIS Tag recombinant protein (fusion Tag composed of 6 histidine residues), flag-Tag recombinant protein (fusion Tag composed of 8 amino acid hydrophilic polypeptide (DYKDDDK)), aviTag recombinant protein (fusion Tag composed of 15 amino acid short peptide), SNAP-Tag recombinant protein (obtained from human O6-methylguanine-DNA methyltransgerase), GST recombinant protein (glutathione-sulfhydryltransferase) Tag, C-Myc Tag recombinant protein (small Tag containing 11 amino acids), etc.
4. The method according to claim 1 a), wherein: the polyelectrolytes include, but are not limited to: proteins, peptides, nucleic acids, inorganic salts, and the like.
5. The method according to claim 1 b), wherein: for the small amount of the hetero electrolyte component, electric field force impurity removal is performed 1 time, wherein when the hetero electrolyte is an amphoteric substance, the PH value of the electrophoresis liquid and the isoelectric Point (PI) of the amphoteric substance in the hetero electrolyte cannot be equal.
6. The method according to claim 1 b), wherein: for the multiple components of the hetero-electrolyte and/or the inclusion of the amphoteric substances, the electric field force is required to remove the impurities for 2 times and/or more than 2 times, and the pH value of the electrophoresis liquid 1 and the pH value of the electrophoresis liquid 2 and/or the pH values of other liquids cannot be equal.
7. The method according to claim 1 b), wherein: when the binding force of the polyelectrolyte and the matrix is large, and/or the concentration of the surfactant or the organic matters in the liquid is increased, the binding force of the polyelectrolyte and the matrix is reduced. And/or increasing the concentration of salt in the liquid and reducing the binding force of impurities with the matrix.
8. The method according to claim 1 b), wherein: the electric field generating the electric field force is a uniform electric field or a non-uniform electric field (electrostatic field).
9. The method according to claim 1 b), wherein: the medium for transmitting the electric field force is electrophoretic fluid or vacuum or thin air.
10. The method according to claim 1 b), wherein: under the action of the electric field force,
-and/or the matrix (diameter D1) bound to the target remains outside the gel (diameter D2), the hetero-electrolyte (diameter D3) entering the gel, wherein D1 > D2 > D3;
-and/or the solid substance (diameter D1) bound to the target remains in the pocket containing a plurality of small holes (pore diameter D4), the polyelectrolyte (diameter D3) moving towards the two poles, wherein D1 > D4 > D3;
and/or the substrate to which the target is bound is attracted to a magnet or a device generating magnetic force, such as an electromagnet or the like, the hetero-electrolyte being moved towards the two poles.
11. The method according to claim 1 b), wherein: voltage: 50V-30000V; current flow: 0mA and/or 10 mA-10A.
12. The substrate and target according to claim 1 b), and/or the substrate and target are washed to further remove the parasitic electrolyte and adsorbed thereon.
13. The method according to claim 1 c), wherein: when the target is an active protein, the separation conditions are as follows:
the liquid cannot contain more than the concentration of denaturing agents that reduce the activity of the biological macromolecules, such as: the liquid does not contain Sodium Dodecyl Sulfate (SDS), or the content thereof is extremely low;
-and/or the liquid contains protein inhibitors such as: phenylmethylsulfonyl fluoride (phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluorid, PMSF), pepstatin, leupeptin (leupeptin), trypsin inhibitor (aprotinin), mixed protease inhibitor, EDTA, disodium edetate, and the like;
-and/or the liquid contains a protein protecting agent, such as: the monohydric alcohol is methanol or ethanol, the dihydric alcohol is ethylene glycol, propylene glycol and butanediol, the trihydric alcohol is glycerol, the glycol polymer is polyethylene glycol, polypropylene glycol and a copolymer of ethylene glycol and propylene glycol, the polyhydroxy monosaccharide is glucose, fructose, mannose and xylose, the disaccharide is sucrose, lactose and maltose, and the polysaccharide is starch, glucan and the like. Preferred is glycerol;
-and/or the temperature is controlled between 0 ℃ and 25 ℃, most preferably at 4 ℃;
and/or absorbing the polyelectrolyte by agarose gel or non-denaturing PAGE or the like.
14. The method according to claim 1 d), wherein: the separation method can be applied in the following experiments including but not limited to: pulldown experiments, immunoprecipitation experiments (IP), co-Immunoprecipitation experiments (Co-Immunoprecipitation, co-IP), chromatin Immunoprecipitation (Chromatin Immunoprecipitation, chIP), RNA-binding protein Immunoprecipitation (RNABinding Protein Immunoprecipitation, RIP), and the like.
15. A label-containing target according to claim 3, including but not limited to: glycoprotein or glycopeptide, phosphorylated protein or phosphorylated peptide, nucleic acid (DNA, RNA, etc.), sugar (monosaccharide, oligosaccharide, polysaccharide, etc.), ubiquitin or ubiquitinated protein, protein with antibody, HIS Tag recombinant protein (fusion Tag composed of 6 histidine residues), flag-Tag recombinant protein (fusion Tag composed of 8 amino acid hydrophilic polypeptide (dykdddk)), aviTag recombinant protein (fusion Tag composed of 15 amino acid short peptide), SNAP-Tag recombinant protein (obtained from human O6-methylguanine-DNA methyltransgerase), GST recombinant protein (glutathione sulfhydryl transferase) Tag, C-Myc Tag recombinant protein (small Tag containing 11 amino acids), and the like.
16. The method according to claim 9, wherein the electrophoresis liquid used is characterized in that:
-at least contain H 2 O；
-and/or contain acids, including but not limited to: hydrochloric acid, glacial acetic acid, sulfuric acid, etc., with hydrochloric acid being preferred;
-and/or contain a base, including but not limited to: sodium hydroxide, tris-base, etc., preferably Tris-base;
-and/or containing salts or salt mixtures, including but not limited to: sodium chloride, sodium acetate, potassium chloride, etc., preferably sodium chloride;
-and/or one or more water-soluble organics, preferably including but not limited to: glycerol, glycine, imidazole, etc., more preferably glycerol;
and/or one or more surfactants, namely cationic, anionic, zwitterionic and/or nonionic. Based on the total volume of the reagent, the dosage of the reagent is as follows: the weight/volume is between 0 and 50%, preferably polyvinylpyrrolidone 40 (PVP 40), 4-nonylphenyl-polyethylene glycol 40 (NP-40), triton X-100 (Triton X-100), etc., and the dosage thereof is as follows: the weight/volume is between 1 and 10 percent;
and/or one or more buffer compounds, preferably having a pH of between 3 and 11, including but not limited to: TRIS (hydroxymethyl) aminomethane (TRIS), N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), N-bis (2-hydroxyethyl) glycine (BICINE), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2- (N-morpholino) ethanesulfonic acid (MES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffers, sodium acetate buffers, methyl acetate buffers, and the like, preferably TRIS, phosphate buffers, sodium acetate buffers, methyl acetate buffers, and the like, at a concentration of: 1 mM-10 mM;
-and/or one or more chelating compounds, preferably selected from the group comprising, but not limited to: N-acetyl-L-cysteine, ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), ethylenediamine-N, N ' -disuccinic acid (EDDS), 1, 2-bis (o-aminophenoxy) ethane-N, N, N ', N ' -tetraacetic acid (BAPTA), and phosphonate chelating agents (including, for example, but not limited to nitrilotris (methylene) phosphonic acid (NTMP), ethylenediamine tetra (methylene phosphonic acid) (EDTMP), diethylenetriamine penta (methylene) phosphonic acid (DTPMP), 1-hydroxyethylidene-1, 1-diphosphine (HEDP), and the like). Preferred are EDTA, DTPA, at concentrations of: 5 mM-100 mM;
-and/or one and/or more RNase a inhibitor mixture, such as: including but not limited to some of the reducing agents disclosed in U.S. patent No. 6825340 or U.S. patent No. 677720. In some embodiments, the commonly used RNase a inhibitor is diethyl pyrocarbonate (DEPC). Preferably 2-mercaptoethanol, 8-hydroxyquinoline, DEPC, etc.
17. The method according to claims 8,9 and 10, characterized in that: one and/or more of the cases taken from claims 8,9 and 10 can be combined to form a new method for removing the polyelectrolyte.
18. The method according to claim 10, wherein: glues include, but are not limited to: agarose gel, denatured polyacrylamide gel (SDS-FAGE), non-denatured polyacrylamide gel (PAGE), and the like.
19. The cleaning solution of claim 12, wherein the composition of the cleaning solution is characterized by:
-at least contain H 2 O；
-and/or contain acids, including but not limited to: hydrochloric acid, glacial acetic acid, sulfuric acid, etc., with hydrochloric acid being preferred;
-and/or contain a base, including but not limited to: sodium hydroxide, tris-base, etc., preferably Tris-base;
-and/or containing salts or salt mixtures, including but not limited to: sodium chloride, sodium acetate, potassium chloride, etc., preferably sodium chloride;
and/or one or more water-soluble organic substances, preferably selected from the group comprising, but not limited to: glycerol, glycine, imidazole, and the like, with glycerol being preferred;
and/or one or more surfactants, namely cationic, anionic, zwitterionic and/or nonionic. Based on the total volume of the reagent, the dosage of the reagent is as follows: weight/volume between 0 and 50%, preferably PVP40, NP-4), triton X-100, etc., in an amount of: the weight/volume is between 1 and 10 percent;
And/or one or more buffer compounds, the pH of which may be between 3 and 11, preferably chosen from, but not limited to: TRIS, TRICINE, BICINE, HEPES, PIPES, CHES, MES, MOPS and/or phosphate buffer, sodium acetate buffer, methyl acetate buffer, etc., preferably TRIS, phosphate buffer, sodium acetate buffer, methyl acetate buffer, etc., at a concentration of: 1 mM-10 mM;
-and/or one or more chelating compounds, preferably selected from the group comprising, but not limited to: N-acetyl-L-cysteine, EDTA, DTPA, EDDS, BAPTA, and phosphonate chelators (including, but not limited to NTMP, EDTMP, DTPMP, HEDP, etc., for example). Preferred are EDTA, DTPA, at concentrations of: 5 mM-100 mM;
-and/or one and/or more RNase a inhibitor mixture, such as: including but not limited to some of the reducing agents disclosed in U.S. patent No. 6825340 or U.S. patent No. 677720. In some embodiments, the commonly used RNase a inhibitor is DEPC. 2-mercaptoethanol, 8-hydroxyquinoline, DEP, and the like are preferable.
20. The isolated active protein of claim 13, comprising: acidic proteins, neutral proteins, and basic proteins.
21. The agarose gel according to claim 18, more particularly 0.5% to 2%, more preferably 1% agarose; more specifically, 8-15% SDS-PAGE, and even more preferably 10-12% SDS-PAGE; more specifically, 8 to 15% of non-denaturing SDS-PAGE, more preferably 10 to 12% of non-denaturing SDS-PAGE, and the like.
22. The method according to claims 1-19, characterized in that: the target and matrix complex can be processed in large volumes to remove adsorbed polyelectrolytes.
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| CN202311588573.6A CN117659115A (en) | 2023-11-19 | 2023-11-19 | Method for removing hetero electrolyte by electric field force and application |
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