Method for artificially modifying 3D photocrosslinking probe and fishing Chinese herbal medicine active micromolecular synthetaseTechnical Field
The invention belongs to the field of biological medicine, and in particular relates to a method for intelligent fishing of Chinese herbal medicine active micromolecular synthetase by utilizing an artificial modified 3D material.
Background
Many small molecules derived from traditional Chinese medicines have good and unique activity, and are one of the important sources for drug development. The traditional method for obtaining natural products of plants is plant extraction, and the traditional production mode has a plurality of defects such as low content and large difference in hosts, long growth cycle of plants per se, and the mode is seriously dependent on the acquisition and consumption of plant resources. In the chemical synthesis, there are problems of long synthetic route, low overall yield, poor economy, etc., and the organic reagents used in the chemical synthesis are easy to cause pollution, and some reactions require expensive heavy metal catalysts, etc., so that industrialization is difficult to realize.
The rare active ingredients of rare plants are rapidly and efficiently obtained in microbial cells through a synthetic biological technology, and a brand new and efficient way is opened up for research and development of natural products of plant sources, reduction of the production cost of medicaments and sustainable utilization and development of plant resources. Successful application of synthetic biology must be based on an in-depth understanding of the biosynthetic pathways of natural products. Compared with microorganisms, due to the huge genome, the biosynthetic gene clusters are not linked, and the like, the biosynthesis analysis of natural products of plant origin is very difficult, and the synthetic routes of some famous molecules with great commercial value, such as taxol, artemisinin and the like, are not completely analyzed so far. The main reason for this is the lack of an effective method for discovering novel enzymes that catalyze unique building block formation reactions in active natural products. Therefore, a set of effective and brand-new strategies are developed to locate the enzyme for synthesizing the natural product in the plant, and the method has great research significance.
The drug target is a biological macromolecule which has a drug effect function in vivo and can be acted by drugs, and most of the drug targets are proteins. Including various receptors, enzymes, etc. The interaction between small molecules and their targets of action is a property that is widely used to identify small molecule protein targets. The traditional chemical biological method is used for identifying the target point of the protein, the main strategy is that the target point is chemically derived through small molecules, the target point is covalently coupled to a specific matrix through chemical bonds, and the protein target point can be enriched on the matrix for fixing the small molecules due to the interaction between the small molecules and the protein target. However, such single site derivatization often results in the loss of activity of the compound, which results in failure of the target identification assay. The interaction of the enzyme with the small molecule substrate or product is dynamic and the completely unknown mode of action of the enzyme with the small molecule is also completely unknown, and if strategies are used for site-directed position-derived small molecule substrates or products, the probability of successful acquisition of the enzyme catalyzing the reaction will be relatively low.
It is reported in literature that Osada and its co-workers in Japan research institute fix high reactive biaziridine group on polymer filler, and under 365nm ultraviolet irradiation, produced carbene can be almost randomly inserted into O/N/S-H and C-H bond to connect with adjacent small molecular structure, thus small molecule can be fixed on filler in various directions, facilitating identification of protein target point. Through constructing a small molecule array based on a carbene photocrosslinking technology, osada et al detect the specific binding of Biotin, rapamycin and other small molecules with targets or antibodies thereof by using a fluorescence intensity method. However, small molecules tend to be immobilized on a two-dimensional (2D) material surface, which may result in failure of protein identification due to crowded surfaces.
The development of surface chemistry provides a new idea for solving the above problems. Three-dimensional surface chemistry has provided plentiful room for improvement in recent years for biomolecular interactions detection chips. Three-dimensional surface chemistry, such as dextran surface chemistry, hydrogel surface chemistry, surface initiated polymerization surface chemistry, and the like, provides a large number of fixing sites for fixing small molecules, expands interaction detection space, and simultaneously improves the nonspecific adsorption resistance of the surface to analytes to a great extent. Wherein, atom Transfer Radical Polymerization (ATRP) is to take simple organic halide as initiator and transition metal complex as halogen atom carrier, and to establish reversible dynamic balance between active species and dormant species through oxidation-reduction reaction, thereby realizing control of polymerization reaction.
Recently, there is a report in literature that the university of john hopkins Liu Jun teaches the subject group that a high throughput screening small molecule microarray was synthesized using ATRP polymerization, and the ability of 2D and newly developed 3D surfaces to bind FKBP12 under the same conditions was compared, and the result shows that the signal background ratio of FKBP12 binding on the 3D surface is 6 times greater than that on the 2D surface, indicating that the newly developed 3D chip is an excellent platform for screening target proteins. And using this 3D microarray to find an effective glucose transporter inhibitor RgA.
Therefore, we can refer to the method to fix the small molecular substrate or product on the surface of the artificial modified three-dimensional polymer material, and the method is used for identifying the enzyme of the plant source catalyzing the specific reaction, and a brand new method for fishing the active small molecular synthetase of the Chinese herbal medicine is developed.
Disclosure of Invention
The invention aims at solving the problems existing in the prior art and provides a method for intelligently fishing active small molecule synthetase of Chinese herbal medicine by using artificial modified 3D materials. The invention not only can screen out the protein target spot of known active small molecules in complex protein background, but also can try to identify the unknown protein target spot of the active small molecules of Chinese herbal medicine, thus laying a solid foundation for finally realizing the microbial synthesis of the active molecules of Chinese herbal medicine.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
An artificial modified 3D photocrosslinking probe takes nano microspheres as a matrix and is modified with the following groups:
where R is a photocrosslinking functional group, m=2-15, n=2-15, preferably, m=5-10, n=5-10.
The photocrosslinking functional group is a benzophenone, aryl azide or bisaziridine group, preferably a trifluoromethyl-phenyl bisaziridine group.
The preparation method comprises the following steps:
A. The nanometer microsphere modified with carboxyl terminal is connected with ethylenediamine; the carboxyl terminal is carboxyl, carboxymethyl or carboxymethoxy; the nano-microsphere is agarose resin, ferroferric oxide nano-microsphere or gold nano-microsphere;
B. Performing amide condensation reaction on the product obtained in the step A and halogenated isobutyric acid, and modifying an initiator group-NH-CO-C (CH2)2 X on the nano-microsphere to obtain the nano-microsphere with the initiator, wherein X is Cl or Br, preferably Br;
C. the nanometer microsphere with initiator and compound hydroxy acrylic ester and polyethylene glycol vinyl monomer are subjected to surface polymerization reaction to obtain 3D material with hydroxyl end, which isModified nanomicrospheres: Wherein m=2-15, n=2-15, preferably m=5-10, n=5-10;
D. Reacting the 3D material with hydroxyl end in the step C with succinic anhydride and 4-dimethylaminopyridine, and connecting carboxyl to the 3D material to obtainModified nano-microspheres;
E. And D, activating the carboxyl of the product in the step D, and reacting with a photocrosslinking agent to finally obtain the artificially modified 3D photocrosslinking probe.
Specifically, in the step A, the nano microsphere modified with carboxyl terminal and ethylenediamine with protecting groupPerforming an amide condensation reaction, and removing a protecting group to obtain a nanometer microsphere modified by-O-CH2CONHCH2CH2NH2; with protecting groups is ethylene diamine of (A)Preferably, fmoc-nonafluorenylmethoxycarbonyl is removed with piperidine to give
The condensing agent used in the amide condensation reaction described in step A, B is preferably a mixture of O-benzotriazole-tetramethylurea Hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole (HOBT) and N, N-Diisopropylethylamine (DIEA) in a molar ratio of 1:0.9-1.2:2.5-3.5, preferably 1:1:3, N-dimethylformamide is taken as a solvent.
The surface-initiated polymerization reaction in the step C is to synthesize an initiator on the surface of the nano microsphere, initiate the monomer to polymerize on the surface through oxidation-reduction reaction, thereby obtaining a so-called 3D structure, and finally connect the photo-crosslinkable group to the surface of the nano microsphere.
In the step C, the surface polymerization reaction comprises the following steps:
And B, carrying out monomer polymerization reaction on the nano microsphere with the initiator obtained in the step B and a solution of vinyl monomers containing an organic reducing agent, a polymerization catalyst, hydroxy acrylic ester and PEG in an anaerobic environment.
Preferably, the organic reducing agent is glucose, ascorbic acid or stannous octoate or dithiothreitol or tris (2-carboxyethyl) phosphine, further preferably the organic reducing agent is present in an amount of 50mM-100mM, preferably 100mM.
Preferably, the polymerization catalyst is a mixture of a transition metal salt, preferably an iron or copper salt, and a chelating agent; the chelating agent is preferably bipyridine, further preferably the transition metal salt is present in an amount of 5mM-10mM, preferably 5mM; the chelating agent is present in an amount of 10mM-20mM, preferably 10mM.
Preferably, the hydroxy acrylic ester monomer is hydroxyethyl methacrylate or hydroxypropyl acrylate or 4-hydroxybutyl acrylate, and the molecular weight is 400-600, preferably 500; the PEG vinyl monomer is polyethylene glycol methacrylate with molecular weight of 300-400, preferably 360, and more preferably, the content of the PEG vinyl monomer is 100mM-1M, preferably 1M.
Preferably, the time for polymerization of the monomer, i.e. polymer growth, is from 6 to 20 hours, preferably 12 hours.
Preferably, the photocrosslinker in step E is selected from the group consisting of benzophenone, aryl azide or bisaziridines, preferably trifluoromethyl-phenyl bisaziridine.
Preferably, step E specifically includes: c, activating carboxyl groups of the nano-microspheres treated in the step C by using NHS and EDCl, adding a solution containing a photocrosslinker and an organic base catalyst for reaction, and then adding a carboxyl blocking agent.
More preferably, the nanoparticle is activated with a DMF solution containing NHS and EDCI; the activation time is 20-40min, preferably 30min; the reaction time with the photocrosslinker and the organic base is 4 to 12 hours, preferably 4 hours; the carboxyl blocking agent is added and reacted for 1 to 6 hours, preferably 2 hours.
Further preferably, the solution containing the photocrosslinker and the organic base catalyst is an organic solvent selected from acetonitrile, tetrahydrofuran and N, N-dimethylformamide.
Further preferably, the organic base catalyst is selected from the group consisting of 4-dimethylaminopyridine, N-diisopropylethylamine and triethylamine.
Further preferably, in the solution, the molar ratio of the terminal amino group of the photocrosslinker to the organic base catalyst is 1: (1-10), further preferably 1:5.
Further preferably, the carboxyl blocking agent is a carboxyl for blocking incomplete reaction on the nanoparticle, preferably ethanolamine.
According to the method, the initiator is modified on the surface of the nano microsphere, the monomer is added, and different numbers of chains with a certain length are synthesized on the nano microsphere through Atom Transfer Radical Polymerization (ATRP), so that a 3D structure is formed, the fixed site of a small molecule can be increased, the success rate of fishing a protein target is improved, and the target protein is more accurately determined. Further connects photo-crosslinking functional group to connect with active small molecule for directly fishing Chinese herbal medicine active small molecule synthetase.
The artificial modified 3D photocrosslinking probe can be used for fishing Chinese herbal medicine active micromolecular synthetase.
A method for fishing Chinese herbal medicine active small molecule synthetase comprises the following steps:
(1) Taking the artificial modified 3D photocrosslinking probe, and connecting the Chinese herbal medicine active small molecules with the artificial modified 3D photocrosslinking probe through photocrosslinking to obtain a 3D probe;
(2) Incubating the plant source total protein disruption solution with a 3D probe, and fishing a small molecule synthetase, namely a protein target;
(3) After incubation, washing the 3D probe after protein target fishing;
(4) Proteins are detected and characterized.
The Chinese herbal medicine active small molecules in the step (1) are preferably small molecules with medicinal value.
In the step (1), the photo-crosslinking conditions are: irradiating with ultraviolet with intensity of 10000-100000uw/cm2, preferably 50000-100000uw/cm2; the irradiation is carried out for 5 to 30 minutes, preferably 15 minutes, under ultraviolet light having a wavelength of 300 to 400nm, preferably 365 nm.
The total protein disruption solution in the step (2) is a total protein disruption solution of plants or a natural protein mixed solution, the concentration of protein in the total protein disruption solution is 1-5mg/ml, and preferably, the natural protein mixed solution is a cell lysis solution.
In the step (3), a solution or a solvent which does not influence the specific binding of the small molecules and the target proteins is used for cleaning, wherein the solution or the solvent comprises an organic solvent or a buffer solution; the organic solvent is preferably tetrahydrofuran, acetonitrile or dimethyl sulfoxide, and the buffer solution is phosphate aqueous solution or Tris buffer solution.
Preferably, the detection and the characterization in the step (4) are that the 3D probe after the protein target is fished is subjected to SDS polyacrylamide gel electrophoresis and silver staining to realize the characterization of the target protein. Further, specific protein bands are taken for subsequent mass spectrometry verification.
The intelligent fishing Chinese herbal medicine active small molecule synthetase disclosed by the invention refers to a process of screening target proteins from a protein mixed solution by manually modifying the interaction between active small molecules on a 3D material and the target proteins.
In a specific embodiment, the method of obtaining a total protein disruption solution of a plant comprises:
(a) Taking frozen plant tissue (such as flower or root), adding cross-linked polyvinylpyrrolidone PVPP or 2-pyrrolidone or polyvinylpyrrolidone, and quick freezing with liquid nitrogen;
(b) Grinding, adding buffer solution and protease inhibitor, dissolving, suspending, ultrasonic crushing, centrifuging, and collecting supernatant;
(c) Adding macroporous exchange resin, shaking, centrifuging, and collecting supernatant.
Preferably, in the step (a), the plant tissue is frozen at the temperature of between 70 ℃ below zero and 80 ℃ below zero, and the mass ratio of the plant tissue to the crosslinked polyvinylpyrrolidone PVPP or 2-pyrrolidone or polyvinylpyrrolidone is 1 to 3:1, preferably 2:1.
In the step (b), grinding is carried out by a tissue grinder, and powder is scraped out; the grinding time is 5min, and the grinding conditions are as follows: 50Hz,60s once; the ultrasonic crushing conditions are as follows: 1200Hz,10min; adding a buffer containing a protease inhibitor; the buffer is 50mmol/L of Tirs buffer with pH=7.4, and contains 100mmol/L NaCl, 10wt% glycerol, 5mmol/L MgCl2、1mmol/L CaCl2, 0.2% NP40.
The macroporous exchange value in the step (c) is Amberlite XAD series macroporous exchange resin, such as Amberlite XAD-4 or Amberlite XAD-2; the shaking time is 20-35min, preferably 30min. The supernatant was centrifuged and the concentration was measured by the Bradford method.
Compared with the prior art, the invention has the following beneficial effects:
The method can utilize the artificial modified 3D material to intelligently fish the synthetase, namely the protein target, of the active micromolecule of the Chinese herbal medicine. The invention not only can screen out the protein target spot of known active small molecules in complex protein background, but also can try to identify the unknown protein target spot of the active small molecules of Chinese herbal medicine, thus laying a solid foundation for finally realizing the microbial synthesis of the active molecules of Chinese herbal medicine. Compared with the traditional method for identifying protein targets by chemical biology, the invention synthesizes the artificially modified 3D photocrosslinking probe, greatly expands the derivative sites of small molecules, and enables the small molecules to be randomly fixed on 3D materials, thereby enhancing the success rate of fishing active small molecule protein targets and more accurately determining target proteins.
Drawings
FIG. 1 is a schematic representation of the fishing of its protein targets in a complex protein background using artificially modified cyclosporin A3D material in example 1 of the present invention.
FIG. 2 is a schematic diagram of the fishing of protein targets from the total protein disruption solution of Artemisia annua by using artificially modified 3D materials of artemisinin, artemisinic acid and dihydroartemisinic acid in example 2 of the present invention.
Fig. 3 is a schematic diagram of fishing a protein target in a total protein crushing liquid of radix salviae miltiorrhizae by using artificially modified salvianolic acid B and rosmarinic acid 3D materials in embodiment 3 of the invention.
Detailed Description
The technical solutions of the present invention will be further described in detail below with reference to specific embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
In this embodiment, the intelligent fishing of cyclosporine a protein target using artificial modified 3D material comprises the steps of:
(1) The synthetic method of the artificial modified 3D material based on the small molecule cyclosporine A comprises the following specific steps:
A. Taking agarose resin based on carboxymethoxy (-O-CH2 COOH)Adding 3 times of HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), 3 times of HOBT (1-hydroxybenzotriazole) and 9 times of DIEA (N, N-diisopropylethylamine) according to the molar ratio, and finally adding 10 times of compoundAnd (3) reacting overnight with DMF as a solvent to obtain the-O-CH2CONHCH2CH2 NH-Fmoc modified nano microsphere: compounds of formula (I)
Then removing Fmoc with 20% piperidine in DMF, oscillating for 30min, washing the microsphere with DMF and methanol, detecting with 2% tetrachlorobenzoquinone and 2% acetaldehyde reagent, and reacting microsphere beads to show blue-green color to obtain the-O-CH2CONHCH2CH2NH2 modified nanometer microsphere:
B. The compound obtained in the step A is mixed with 10 times of 2-bromoisobutyric acid, 3 times of HBTU, 3 times of HOBT and 9 times of DIEA according to the molar ratio, the mixture is reacted for 4 hours, the microspheres are washed by DMF and MEOH, and finally the microspheres are detected by 2% of chloranil and 2% of acetaldehyde reagent, and the microspheres do not develop color. Finally, the microsphere connected with the initiator group is obtained:
C. According to the principle of atom transfer radical polymerization reaction initiated by the surface, 50 times of CuBr and 100 times of Bpy (bipyridine) are added into a reaction bottle firstly, the gas is pumped and exchanged, the microsphere obtained in the step B is added under the anaerobic condition, 2500 times of compound hydroxyethyl methacrylate and 10000 times of polyethylene glycol methacrylate are added, and finally 1000 times of ascorbic acid is added for reaction overnight; the next day the microspheres were washed with methanol. The reaction solvent is methanol: water (volume ratio) =1: 1, a step of; obtaining the 3D materialWherein m, n=5-10;
D. Reacting the 3D material obtained in the step C with 5eq succinic anhydride and 3eq 4-dimethylaminopyridine, using DMF as solvent for 2 hours, and washing with DMF for 3 times to obtain the compound
E. the compound obtained in step D was reacted with 10eq NHS, 10eq EDCI and 20eq DIEA in DMF solution for 30min to activate the carboxyl group. After the activation is finished, adding 20eq DIEA and 5eq trifluoromethyl-phenyl bisaziridine serving as a photo-crosslinking agent, reacting in the dark for 4 hours, adding 1M ethanolamine solution immediately after the reaction is finished, continuing the reaction for 2 hours, adding ethanolamine to seal unreacted activated carboxyl groups, and finally obtaining the artificially modified 3D photo-crosslinking probe;
F. And thoroughly cleaning the artificially modified 3D photocrosslinking probe by using DMF and tetrahydrofuran after the end of the sealing, transferring the 3D photocrosslinking probe to a 4ml glass sample bottle, adding a cyclosporin A micromolecular solution with 1 time of molar weight according to the quantity of activated carboxyl, spin-drying, drying by N2, irradiating for 15min by using 365nm ultraviolet light in a dry state, and cleaning beads by using tetrahydrofuran, DMSO and PBS to obtain the final cyclosporin A artificially modified 3D material.
(2) Incubating the cyclosporine A artificially modified 3D material prepared in the step (1) with a jurkat-T cell lysate for 40 minutes. The preparation method of the jurkat-T cell lysate is as follows: collecting two large disks of jurkat-T cells, centrifuging at 1000rpm and 4 ℃ for 10min, discarding the supernatant, re-spinning with 2ml buffer (50 mM Tris, PH=7.40, 100mM NaCl, 10% glycerol, 5mM MgCl2、1mM CaCl2, 0.2% NP40 and protease inhibitor Protease Inhibitor), performing ultrasonic treatment for 10min (stopping 50s after 10s ultrasonic treatment, power 100w and total 10 min), centrifuging at 12000rmp at 4 ℃ for 10min, and taking the supernatant as jurkat-T cell lysate, wherein the concentration of the cell lysate is at least 3mg/ml measured by the Bradford method. After the incubation is completed, the 3D agarose resin is washed by the buffer for 3 times;
(3) And (3) adding 50ul of 5-x SDS loading buffer solution, heating for 5min at 100 ℃, centrifuging, performing SDS-PAGE gel electrophoresis, and performing silver staining on the resin after the washing in the step (2).
FIG. 1 is a schematic diagram of the fishing of its protein targets in a complex protein background using an artificially modified cyclosporin A3D material. From the figure, it can be seen that the artificially modified cyclosporin A3D material prepared by us successfully hooks the protein targets cyclophilins A and B (CypA and CypB) in the jurkat-T cell lysate. In addition, the experimental group had few other distinct hetero protein bands, indicating that this method is a specific binding target fishing. Blank refers to 3D material without cyclosporine a small molecule added.
Eluting the target protein from the resin washed in the step (2), namely, synthesizing enzyme related to the synthesis of cyclosporin A, and further researching.
Example 2
In this embodiment, the artificial modification of 3D material is used to intelligently fish possible protein targets of arteannuic acid, arteannuic acid and dihydroarteannuic acid, comprising the steps of:
(1) Step (1) of example 1 is followed except that the cyclosporin A small molecule solution of step F is replaced with arteannuic acid or dihydroarteannuic acid solution.
(2) Incubating the small molecule artificial modified 3D material prepared in the step (1) with the artemisia annua total protein disruption solution for 40 minutes. The preparation method of the artemisia annua total protein crushing liquid comprises the following steps: 3g of artemisia annua flowers frozen at the temperature of minus 80 ℃ are taken, 1.5 g of crosslinked polyvinylpyrrolidone PVPP is added, quick freezing is carried out by using liquid nitrogen, a tissue grinding instrument is used for grinding for 5min (50 Hz,60s are used for once), powder is scraped out, the powder is dissolved and resuspended by using 5ml buffer(50mm Tris(PH＝7.40),100mM NaCl 10％GLYCEROL,5mM MgCl2,1mM CaCl2,0.2％NP40,Protease Inhibitor),, ultrasonic crushing is carried out for 10min (120 Hz), the supernatant is centrifugally taken, 1.5 g of ion exchange resin Amberlite XAD-4 is added into the supernatant, shaking is carried out for 30min, the supernatant is centrifugally taken, the concentration is measured by using the Bradford method, and the concentration of the total protein crushing liquid of the artemisia annua is at least 2mg/ml. After incubation was completed, 3D agarose resin was washed 3 times with the buffer described above (without 0.2% np40 and protease inhibitor);
(3) And (3) adding 50ul of 5-x SDS loading buffer solution, heating for 5min at 100 ℃, centrifuging, performing SDS-PAGE gel electrophoresis, and performing silver staining on the resin after the washing in the step (2).
FIG. 2 is a schematic diagram of the fishing of protein targets from the total protein disruption solution of Artemisia annua by using artificially modified 3D materials of artemisinin, artemisinic acid and dihydroartemisinic acid in example 2 of the present invention. From the figure it can be seen that there are distinct protein bands around 35kd, 27kd and 10kd, which are most likely proteins that bind to artemisinin or arteannuin.
Example 3
In this embodiment, the method for intelligently fishing possible protein targets of salvianolic acid B and rosmarinic acid by using the artificial modified 3D material comprises the following steps:
(1) The same as in (1) of example 1, except that the cyclosporin a small molecule solution of step F was replaced with salvianolic acid B or rosmarinic acid solution.
(2) Incubating the small molecule artificial modified 3D material prepared in the step (1) with the total protein disruption solution of the root of red-rooted salvia for 40 minutes. Root of red sage total protein crushing the preparation method of the liquid comprises the following steps: taking 4 g of red sage root frozen at the temperature of minus 80 ℃, adding 2 g of crosslinked polyvinylpyrrolidone PVPP, quick freezing by liquid nitrogen, grinding by a tissue grinding instrument for 5min (50 Hz,60s for one time), scraping out powder, dissolving by 6ml buffer(50mm Tris(PH＝7.40),100mM NaCl 10％GLYCEROL,5mM MgCl2,1mM CaCl2,0.2％NP40,Protease Inhibitor),, re-suspending by ultrasound, crushing for 10min (120 Hz), centrifuging to obtain a supernatant, adding 2 g of ion exchange resin Amberlite XAD-4 into the supernatant, oscillating for 30min, centrifuging to obtain the supernatant, measuring the concentration by a Bradford method, wherein the concentration of the total protein crushing liquid of the red sage root is at least 0.8mg/ml. After incubation was completed, 3D agarose resin was washed 3 times with the buffer described above (without 0.2% np40 and protease inhibitor);
(3) And (3) adding 50ul of 5-x SDS loading buffer solution, heating for 5min at 100 ℃, centrifuging, performing SDS-PAGE gel electrophoresis, and performing silver staining on the resin after the washing in the step (2).
Fig. 3 is a schematic diagram of fishing a protein target in a total protein crushing liquid of radix salviae miltiorrhizae by using artificially modified salvianolic acid B and rosmarinic acid 3D materials in embodiment 3 of the invention. As can be seen from the figure, there are distinct protein bands around 50kd, 25kd and 16kd, which are likely to be proteins that bind to salvianolic acid B or rosmarinic acid.
The applicant states that the present invention is described by way of the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be practiced in dependence on the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.