CN110627864A - Automatic extraction method of cell components - Google Patents
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- CN110627864A CN110627864A CN201911082471.0A CN201911082471A CN110627864A CN 110627864 A CN110627864 A CN 110627864A CN 201911082471 A CN201911082471 A CN 201911082471A CN 110627864 A CN110627864 A CN 110627864A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M45/00—Means for pre-treatment of biological substances
- C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
Abstract
The invention belongs to the field of biotechnology, and discloses an automatic extraction method of cell components, which comprises the following steps: (1) mixing the cell sample and the sensitizer, and adding the mixture into a sample adding groove for incubation; (2) after incubation for 5-10min, a pipettor added the sample to the filter tube; (3) air injection pressurization, wherein cells are crushed under the action of shearing force and inertial impact; (4) if the total cellular protein or the protein with a special distribution of a certain subcellular structure needs to be collected, the product obtained in the step (3) can be pretreated firstly, and then a series of cell component extracts are added into the filtered product for extraction. The method is different from the traditional method which completely depends on the chemical crushing of the lysate or the grinding and homogenizing method for crushing, so that the cell crushing is more uniform, the efficiency is high, and the protein damage is small. The method is suitable for processing with small number of samples and batch processing under the condition of large total number of samples, has small requirement on single sample amount, can effectively save manpower and avoid the difference of manual operation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a device and a method capable of automatically separating proteins and subcellular structures in animal and plant cells, which are suitable for batch processing of multiple samples.
Background
In the post-genome era, the development of functional omics is the main content of life science research, and proteins are the main executives of life activities and thus the focus of research. By analyzing the similarities and differences of proteins in tissues, cells or subcellular structures under different physiological/pathological conditions, the complex life phenomena are revealed, and targets are found for disease diagnosis and treatment. Due to the wide variety of proteins (more than 10 ten thousand) in a cell, the dynamic distribution range of different proteins is very wide (from 10)2To 109Individual molecules) and vary in nature, which cause difficulties in the isolation and analysis of proteins. Some pre-separation means are adopted to separate protein from non-protein biomacromolecule, so as to reduce sample complexity, and the method is a basic method for analyzing and researching specific protein. With the progress and refinement of life science research, the research stage of proteins with subcellular structure specific distribution has been entered, and therefore, the separation of proteins with subcellular specific distribution is a prerequisite for subcellular proteomics research.
The traditional method for separating cell protein is to adopt RIPA lysate (the main components are 50mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS) to lyse cells, the protein is dissolved in the lysate, the insoluble part is discarded after high-speed centrifugation by a centrifugal machine, the supernatant is the separated protein solution, the protein which can be separated by the method must be dissolved by the cell lysate, about 30% of the protein is insoluble in the RIPA lysate in fact, the protein exists in the centrifugal precipitate and is discarded, and therefore the target protein is lost or the protein quantification is inaccurate. The classical method for separating subcellular structures is to perform separation by combining differential centrifugation and density gradient centrifugation after cell lysis homogenization, and the method needs to be provided with a copied density gradient medium and an ultra-high speed centrifuge, so that the method has high requirements on experimental skills and experimental equipment of researchers, and has the disadvantages of complex operation, large sample quantity requirement, long time consumption and poor repeatability. In recent years, several methods for separating subcellular based on antibody affinity have been developed, which utilize the specificity of immunoaffinity, so that it is necessary to prepare a specific antibody for each structure, and although the specificity is improved, the antibody is expensive and the experiment cost is too high. Meanwhile, because a large amount of RIPA is used for cell lysis and protein lysis in the traditional method, the contained anionic surfactants such as SDS and the like can be efficiently lysed, but the protein can be lost and denatured. The traditional physical cell disruption means is ultrasonic or glass homogenizer homogenization, the ultrasonic method is not suitable for scientific research with small sample amount, and the thermal effect of the ultrasonic can cause protein denaturation; the homogenization effect is artificially large, insufficient homogenization effect or excessive homogenization is easy to occur, subcellular structures are damaged, and downstream separation is influenced. Meanwhile, homogenate is manually operated, so that the whole low-temperature environment is difficult to ensure, and protein denaturation or degradation is caused. The released genomic nucleic acid simultaneously makes the cell homogenate extremely viscous, which affects further isolation experiments.
In the traditional method, low-temperature high-speed centrifugation and even ultra-high-speed centrifugation are necessary separation methods, the centrifugation operation needs to manually complete sample placement and recovery, and the automation is not suitable for being integrated with other operation links, so that no automatic cell protein extraction device exists at present. Under the requirement of multi-sample separation and extraction, the manual operation has high working strength, the batch difference risk is improved, and the scientificity of experimental results is certainly influenced, so that technical innovation is necessary and an automatic extraction device is developed.
Disclosure of Invention
In order to overcome the defects of centrifugal operation, the invention provides an automatic filtration and extraction method of cell components, which is different from the traditional method that the chemical crushing of lysate is completely relied on or the crushing is carried out by a grinding and homogenizing method, so that the cell crushing is more uniform. The method is suitable for processing with small number of samples, is more suitable for batch processing under the condition of large total number of samples, has small requirement on single sample amount and high processing efficiency, can effectively save manpower, and avoids the difference of manual operation.
The purpose of the invention is realized by the following technical scheme:
an automatic extraction method of cell components, the instrument used in the method comprises an automatic extraction device of cell components, and the used reagent comprises a sensitizing agent and a series of cell protein extraction liquid;
the automatic cell component extraction device includes: the device comprises a liquid transferring system, a filtering system, a cooling system and an automatic control system, wherein the cooling system provides low temperature of 0-4 ℃ for the liquid transferring system and/or the filtering system, and the automatic control system is respectively connected with the liquid transferring system, the filtering system and the cooling system; the filtration system includes: the filter tube placing box, the ventilation and pressure supply system and the filter tube, wherein the filter tube comprises a filter column and a collecting tube, the filter column comprises a filter element, the filter element breaks the cell sensitized by the cell membrane and filters the nucleic acid DNA in the process of compressed gas pressure filtration, and the obtained filtrate is other cell components without DNA; the pipetting system comprises: a pipette tip placing box, a sample adding groove, a pipette and a pipette tip; the pipettors are arranged in rows and are arranged in parallel with the sample adding grooves, the sample adding grooves are sharp-bottom round hole groups communicated in rows, and the number and the positions of the sample adding grooves correspond to the jacks of the pipette tip placing box; the ventilation and pressure supply system comprises a fence, a compressed gas cylinder, a ventilation pipeline and an air nozzle; the compressed gas cylinder is connected with the air nozzle through an air duct, a main valve is arranged between the compressed gas cylinder and the air duct, the air duct is connected with the air nozzle through threads, a branch valve is arranged between the air duct and the air nozzle, and a rubber gasket is arranged at the contact position of the air nozzle and the filter tube;
the sensitizer comprises polyethylene glycol octyl phenyl ether, ethyl phenyl polyethylene glycol and a buffer salt solution;
the series of cell component extracts comprises: polyethylene glycol octyl phenyl ether, ethyl phenyl polyethylene glycol, polyoxyethylene sorbitan monolaurate, and 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt;
the method comprises the following steps:
(1) inserting the suction head into a suction head placing box jack of a pipette, inserting a predetermined number of filter tubes into a filter tube placing box, and finally mixing a certain amount of cell samples and a sensitizer and adding the mixture into a sample adding groove for incubation;
(2) starting an automatic extraction program, after incubation for 5-10min, moving a pipettor to the position above a pipette tip placing box, and downwards moving and inserting a pipette tip; moving the pipettor to the upper part of the sample adding groove, and downwards moving to suck the sample mixed liquid; moving the pipettor to the upper part of the filter tube placing box, and moving downwards to add the sample into the filter tube; moving the pipettor back to the home position;
(3) the ventilation pressure supply system moves downwards, the air nozzle is inserted into the filter tube, the upper edge of the filter tube is tightly pressed and sealed with the rubber gasket, the air nozzle sprays pressurized gas, so that cell suspension treated by the sensitizing agent rapidly passes through the filter element, cells are crushed under the action of shearing force and inertial impact, and the ventilation pressure supply system moves upwards to the original position; taking out the filter tube, and collecting the filtrate in the tube, namely other cell components without DNA;
(4) if the total cell protein or the protein with a certain subcellular structure specially distributed is required to be collected, the product obtained in the step (3) can be centrifuged to obtain a subcellular structure precipitate, then a corresponding series of cell component extract is added, and the product obtained after all insoluble substances are dissolved is the specific protein extract contained in a certain subcellular structure.
Further, the purpose of the invention can be realized by the following technical scheme:
the filter element is a microporous structure made of hydrophobic polyolefin high polymer materials.
The high polymer material comprises polyethylene, polypropylene and polytetrafluoroethylene.
The filter element is processed into particles with the particle size of 10-150 mu m by using a high polymer material, and the particles are sintered into a microporous structure by pressurization, wherein the pore size range is 20-60 mu m, and the thickness range is 1mm-10 mm.
The gas used by the ventilation and pressure supply system is nitrogen, and the cooling system can be a water-cooling heat exchange pipe connected with ice water or a compressor cooling system.
The cell sensitizer comprises 0.10 mass percent of ~ 2.00.00 mass percent of polyethylene glycol octyl phenyl ether, 0.10 mass percent of ~ 2.50.50 mass percent of ethyl phenyl polyethylene glycol and a buffer salt solution, wherein the buffer salt solution is 20-70mM Tris-hydrochloric acid buffer salt solution, has the pH of 6.5-8.0 or is 0.1 time of ~ 3 times of PBS phosphate buffer salt solution, and has the pH of 6.5-8.0.
The series of cell component extracts comprise 0.30 to ~ 2.20.20 mass percent of polyethylene glycol octyl phenyl ether, 0.10 to ~ 4.00.00 mass percent of polyoxyethylene sorbitan monolaurate, 0.20 to ~ 2.10.10 mass percent of ethyl phenyl polyethylene glycol and 0.10 to ~ 3.00.00 mass percent of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, wherein a buffer solution system comprises 100 to 250mM sodium chloride and 20 to 70mM Tris-hydrochloric acid buffer solution, the pH value is 6.5 to 8.0 or 0.1 to ~ 3 times of PBS phosphate buffer solution, and the pH value is 6.5 to 8.0.
Advantageous effects
The invention provides a device for automatically extracting cell protein and other components without DNA from cells, which realizes cell disruption by combining a chemical sensitizer with mechanical action, wherein the mechanical action utilizes the shearing force and the inertia impact action when cell suspension rapidly passes through a porous filtering material under the action of gas pressure, and is different from the traditional method which completely depends on the chemical disruption of lysate or the disruption by a grinding method, so that the cell disruption is more uniform and the efficiency is higher. The method is suitable for processing with small number of samples and batch processing under the condition of large total number of samples, has small requirement on single sample amount, can effectively save manpower and avoid the difference of manual operation.
The filter column with a porous structure plays a role in mechanically breaking cells on one hand, and has a filtering effect on sticky nucleic acid released by cell breaking on the other hand, so that DNA molecules are effectively removed, and proteins and subcellular structures are separated. Compared with the traditional differential centrifugal separation and density gradient centrifugal separation, the method has higher separation speed and does not need complex density gradient media and high-grade equipment.
The cell pretreatment adopts a sensitizer which is different from the traditional lysis solution, the composition changes the type of the surfactant, and the mild nonionic surfactant replaces the traditional ionic surfactant (such as SDS and deoxycholate) to be matched with hypotonic salt solution. The sensitizer can not directly crack cell membranes but sensitize the cell membranes, so that the sensitizer is more sensitive to mechanical force, is beneficial to breaking cells by mechanical force when a sample passes through a filter column, and can reduce the damage to proteins. In the membrane protein separation, the structure of a cell membrane can be maintained to be larger fragments, which is beneficial to further separation; the relative integrity of the organelles is kept, the separation difficulty of subsequent subcellular structures is reduced, and the organelles can be separated without ultracentrifugation.
The optimized cell protein extracting solution has the function of dissolving insoluble protein in filtrate or extracting special protein contained in subcellular structure. Various mild nonionic surfactants are used to replace the traditional single ionic surfactant, so that the range of the dissolved protein is wider and the extraction effect is milder. Unlike conventional RIPA, it consists of a strong anionic surfactant SDS and sodium deoxycholate, and can dissolve part of the protein but easily destroy the higher structure of the protein.
Drawings
FIG. 1 is a schematic structural diagram of an automatic protein extraction apparatus according to the present invention.
Fig. 2 is a schematic structural view of the ventilation and pressure supply system in fig. 1.
FIG. 3 is a schematic view of a filter tube structure according to the present invention.
Fig. 4 is a schematic view of the structure of the collector tube of fig. 3.
Fig. 5 is a schematic view of the filter cartridge structure of fig. 3.
FIG. 6 is a graph showing the results of electrophoresis obtained in example 2 of the present invention.
FIG. 7 is a graph showing the results of electrophoresis obtained in example 3 of the present invention.
FIG. 8 is a diagram showing the result of Western blotting hybridization performed in example 3 of the present invention.
FIG. 9 is a graph showing the results of electrophoresis obtained in example 4 of the present invention.
Reference numbers in the figures: the device comprises a pipette tip placing box 1, a pipette 2, a ventilating and pressure supplying system 6, a filter tube placing box 7, a sample adding groove 8 and a filter tube 9.
Detailed Description
In order to clearly explain the technical features of the present invention, the following detailed description of the present invention is provided with reference to the accompanying drawings. It should be noted that the components illustrated in the figures are not necessarily drawn to scale. Descriptions of well-known components and techniques are omitted so as to not unnecessarily limit the invention. The described embodiments are only some, but not all embodiments of the invention. The embodiments are illustrative, and are not to be construed as limiting, and all other embodiments that can be derived by one of ordinary skill in the art without making any inventive step are intended to be within the scope of the present invention. Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
The following describes embodiments of the present application in further detail with reference to the accompanying drawings.
Example 1
As shown in fig. 1 ~ 3, an apparatus for automatic extraction of cell components comprises an automatic cell component extraction device, and reagents including a sensitizer and a series of cell protein extracts;
the automatic cell component extraction device includes: the device comprises a liquid transferring system, a filtering system, a cooling system and an automatic control system, wherein the cooling system provides low temperature of 0-4 ℃ for the liquid transferring system and/or the filtering system, and the automatic control system is respectively connected with the liquid transferring system, the filtering system and the cooling system; the filtration system includes: the device comprises a filter tube placing box 7, a ventilation and pressure supply system 6 and a filter tube 9, wherein the filter tube 9 comprises a filter column 9-3 and a collecting tube 9-1, the filter column 9-3 comprises a filter element 9-2, the filter element 9-2 breaks cells sensitized by cell membranes in the process of compressed gas pressure filtration and filters nucleic acid DNA, and the obtained filtrate is other cell components without DNA; the upper diameter of the filter column is smaller than the inner diameter of the collecting pipe, and a gap is formed to discharge gas. The filter column with a porous structure plays a role in mechanically breaking cells on one hand, and has a filtering effect on sticky nucleic acid molecules released by cell breaking on the other hand, so that DNA molecules are effectively removed, and proteins and subcellular structures are separated. Compared with the traditional differential centrifugal separation and density gradient centrifugal separation, the method has higher separation speed and does not need complex density gradient media and high-grade equipment; the pipetting system comprises: a pipette tip placing box 1, a sample adding groove 8, a pipette 2 and a pipette tip; the pipettors 2 are arranged in rows and are arranged in parallel with sample adding grooves, the sample adding grooves are sharp-bottom round hole groups communicated in rows, and the number and the positions of the sample adding grooves correspond to the pipette suction head placing box holes; the pipette tip placing box is a slot suitable for a square pipette tip box on the market, and the uncapped pipette tip box can be placed into the slot; the sample adding slot is a group of pointed-bottom round holes which are communicated in rows, the number and the positions of the holes correspond to those of the placing box, the mixed sample of the cells and the reagent can be added from any hole, and the sample amount of each hole is the same through communication. The ventilation pressure supply system 6 comprises a fence 6-1, a compressed gas cylinder 6-4, a ventilation pipeline 6-5 and an air nozzle 6-7; the compressed gas cylinder is connected with the air nozzle through an air pipe, a main valve 6-6 is arranged between the compressed gas cylinder and the air pipe, the air pipe is connected with the air nozzle through threads, a branch valve 6-3 is arranged between the air pipe and the air nozzle, and a rubber gasket 6-2 is arranged at the contact part of the air nozzle 6-7 and the filter tube. The length and width of the peripheral column are slightly larger than the placing box, and the peripheral column is provided with a chamfer with a certain angle so as to ensure that the ventilation pressure supply system is aligned with the placing box in long-term use. The interior of the filter tube is provided with rows of air nozzles, and the air nozzles are provided with rubber gaskets which can realize sealing after being butted with the upper edge of the filter tube. The jet port is connected with the pipeline and the compressed gas cylinder through internal threads, the pipeline connected with each gas port is controlled by an electric control valve, and the gas cylinder end is connected with a main valve, so that the gas cylinder is convenient to replace. The filter tube placing box is composed of row holes in which filter tubes can be placed.
The sensitizer comprises polyethylene glycol octyl phenyl ether, ethyl phenyl polyethylene glycol and a buffer salt solution;
the series of cell component extracts comprises: polyethylene glycol octyl phenyl ether, ethyl phenyl polyethylene glycol, polyoxyethylene sorbitan monolaurate, and 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt;
the method comprises the following steps:
(1) inserting the suction head into a suction head placing box jack of a pipette, inserting a predetermined number of filter tubes into a filter tube placing box, and finally mixing a certain amount of cell samples and a sensitizer and adding the mixture into a sample adding groove for incubation;
(2) starting an automatic extraction program, after incubation for 5-10min, moving a pipettor to the position above a pipette tip placing box, and downwards moving and inserting a pipette tip; moving the pipettor to the upper part of the sample adding groove, and downwards moving to suck the sample mixed liquid; moving the pipettor to the upper part of the filter tube placing box, and moving downwards to add the sample into the filter tube; moving the pipettor back to the home position;
(3) the ventilation pressure supply system moves downwards, the air nozzle is inserted into the filter tube, the upper edge of the filter tube is tightly pressed and sealed with the rubber gasket, the air nozzle sprays pressurized gas, so that cell suspension treated by the sensitizing agent rapidly passes through the filter element, cells are crushed under the action of shearing force and inertial impact, and the ventilation pressure supply system moves upwards to the original position; taking out the filter tube, and collecting the filtrate in the tube, namely other cell components without DNA;
(4) if the total cell protein or the protein with a certain subcellular structure specially distributed is required to be collected, the product obtained in the step (3) can be centrifuged to obtain a subcellular structure precipitate, then a corresponding series of cell component extract is added, and the product obtained after all insoluble substances are dissolved is the specific protein extract contained in a certain subcellular structure.
In a preferred embodiment, the filter element is a microporous structure made of hydrophobic polyolefin polymer material.
The polymer material in the preferred embodiment includes polyethylene, polypropylene, and polytetrafluoroethylene.
Preferably, the filter element of the embodiment is made of polymer material, processed into particles with a particle size of 10-150 μm, and sintered into a microporous structure by pressurization, wherein the pore size ranges from 20 to 60 μm, and the thickness ranges from 1mm to 10 mm.
The gas used by the ventilation and pressure supply system in the preferred embodiment is nitrogen, and the cooling system may be a water-cooled heat exchange pipe connected with ice water or a compressor cooling system.
The cell sensitizer of the embodiment preferably comprises 0.10% ~ 2.00.00% of polyethylene glycol octyl phenyl ether (Triton X-100), 0.10% ~ 2.50.50% of ethyl phenyl polyethylene glycol (NP-40) and a buffer salt solution, wherein the buffer salt solution is 10-250mM of sodium chloride, 1-20mM of magnesium chloride, 20-70mM of Tris-hydrochloric acid buffer salt solution, pH 6.5-8.0 or 0.1 time ~ 3 times of PBS phosphate buffer salt solution, and the pH is 6.5-8.0.
The preferential cell component extract of the series of the embodiment comprises 0.30 percent of ~ 2.20.20 percent of polyethylene glycol octyl phenyl ether, 0.10 percent of ~ 4.00.00 percent of polyoxyethylene sorbitan monolaurate, 0.20 percent of ~ 2.10.10 percent of ethyl phenyl polyethylene glycol, 0.10 percent of ~ 3.00.00 percent of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, a buffer solution system comprises 100 percent of sodium chloride, 20 to 70 percent of Tris-hydrochloric acid buffer solution, 6.5 to 8.0 or 0.1 to ~ 3 times of PBS phosphate buffer solution, and the pH value is 6.5 to 8.0, so that various amphiphilic or hydrophobic insoluble components can be effectively dissolved.
Example 2 extraction of human hepatoma cell Total protein
Collect 5X 10 per well6HepG2 cells were washed with pre-cooled PBS. About 200. mu.L of cell sensitizing solution was added to each well, added to the cell sample, mixed well, and added to the sample addition tank. And putting the corresponding filter tube into the filter tube placing box hole. The filter element is formed by sintering polytetrafluoroethylene particles with the average particle size of 50 mu m, the void ratio is 45%, and the average pore diameter is 30 mu m. Selecting an air jet and a sample adding groove.
Starting an automatic extraction program, incubating at the low temperature of 0-4 ℃ for 5 minutes, moving the row-type pipettor to the position above the pipettor sucker placing box, and moving down and inserting the pipettor sucker. Move to the loading slot, aspirate 200 μ L of cell suspension, then move to the filter tube placement cassette, add to the filter tube, and move the pipettor back into place. The ventilating and pressure supplying system moves downwards, the air nozzle is in butt joint with the filtering column for sealing, and the mixed suspension passes through the filtering column by the load air pressure.
Taking out the collecting pipe, and collecting the filtrate in the pipe to obtain the liver cancer cell total protein extracting solution with the genome DNA removed. The extract is not sticky and can be directly subjected to subsequent analysis. In the traditional method, such as Biyuntian protein extraction kit (product No. P0013B) and Thermoscientific animal cell protein extraction reagent (product No. 78501), the cell lysate contains a protein extract of genomic DNA, and high-speed centrifugation is needed to remove the genomic DNA. After electrophoretic analysis, as shown in FIG. 6, lane 1 is Biyuntian kit, lane 2 is Thermo Scientific kit, and lane 3 is total protein extracted by the method. It can be known that this device draws with supporting total protein extraction kit, compares used commercial kit in laboratory, and protein extraction efficiency is higher, and it is more complete to extract the protein spectrum. And takes a short time of only 10 minutes, which is about 45 minutes for processing one sample by the conventional method.
Example 3: extraction of human liver cancer membrane protein
Collect 5X 10 per well6HepG2 cells were washed with pre-cooled PBS. Adding about 200 mu L of membrane protein sensitizing solution into each hole, adding into the cell sample, mixing, and adding into the sample adding slot. And putting the corresponding filter tube into the filter tube placing box hole. Selecting an air nozzle and a sample adding groove.
Starting an automatic extraction program, incubating at the low temperature of 0-4 ℃ for 10 minutes, moving the row-type liquid transfer device to the position above a liquid transfer device sucker placing box, and moving downwards to insert and take the sucker. Move to the loading slot, aspirate 200. mu.l of the mixed sample, then move to the cartridge for placing the filtration tube, add the sample to the filtration tube, and move the pipettor back to the home position. The ventilating and pressure-supplying system moves downwards, the air nozzle is closed with the filter column, the air nozzle sprays pressurized gas, so that the cell suspension treated by the sensitizer rapidly passes through the filter element, and the cells are crushed under the action of shearing force and inertial impact. The vent pressure supply system moves back into place. And taking out the filter tube, and collecting the filtrate obtained in the tube, namely the total cell component extract without DNA.
The collection tube was removed and placed in a normal centrifuge at 700g for 1 minute. The supernatant was transferred to a new collection tube and centrifuged at 16000g for 30 min. Removing supernatant, adding 100 μ l of membrane protein extract (0.5-1.5 wt% ethylphenylpolyethylene glycol, 0.5-1.5 wt% polyethylene glycol octylphenyl ether, 0.1-0.5 wt% 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, 100 mM sodium chloride, 2-10mM EDTA, 20-70mM Tris, hydrochloric acid to adjust pH to 6.5-8) to the precipitate. Thus obtaining HepG2 cell membrane natural protein solution. Subsequent experimental analysis can be performed directly. Electrophoretic analysis and Western Blot protein immunoblotting analysis (Anti-ATPA 1 is selected as a primary antibody, and whether the membrane protein marker protein-sodium potassium ATP enzyme exists and the content of the membrane protein marker protein-sodium potassium ATP enzyme contained in the extracted membrane protein extracting solution can be evaluated, so that the extraction effect is reflected laterally). FIG. 7 shows the results of electrophoresis: 1. lane 3 is a commercial kit adopting a traditional method, 1 is a Biyuntian membrane protein extraction kit (product number P0033), 3 is a Thermo Scientific animal cell membrane protein extraction reagent (product number 89842), and 2 is the membrane protein extracted by the method; FIG. 8 shows Western blot hybridization results: 1. lane 3 is a commercial kit, and lane 2 is the membrane protein extracted by the method. Compared with a commercial kit used in a laboratory, the device and the matched membrane protein extraction kit have the advantages of high extraction efficiency and complete protein extraction spectrum, and the device and the matched membrane protein extraction kit are small in cell amount and short in time, and the extracted protein is natural protein.
Example 4: extraction of human liver cancer cell mitochondrion protein
Collect 1X 10 per well7HepG2 cells were washed with pre-cooled PBS. About 200. mu.L of cell sensitizing solution was added to each well, added to the cell sample, mixed well, and added to the sample addition tank. And putting the corresponding filter tube into the filter tube placing box hole. The filter element is formed by sintering polytetrafluoroethylene particles with the average particle size of 50 mu m, the void ratio is 45%, and the average pore diameter is 30 mu m. Selecting an air jet and a sample adding groove.
Starting an automatic extraction program, incubating at the low temperature of 0-4 ℃ for 5 minutes, moving the row-type pipettor to the position above the pipettor sucker placing box, and moving down and inserting the pipettor sucker. Move to the loading slot, aspirate 200 μ L of cell suspension, then move to the filter tube placement cassette, add to the filter tube, and move the pipettor back into place. The ventilating and pressure supplying system moves downwards, the air nozzle is in butt joint with the filtering column for sealing, and the mixed suspension passes through the filtering column by the load air pressure.
The collection tube was removed, centrifuged at 700g for 1 minute, the supernatant was aspirated into a new collection tube, 400. mu.l of a mitochondrial classification buffer (cell sensitizer free of surfactant component) was added thereto, centrifuged at 16000g for 20 minutes, the supernatant was removed, and centrifuged at 8000g for five minutes, and 200. mu.l of a mitochondrial classification buffer was added thereto.
The supernatant was added to 1.6ml of precooled PBS and centrifuged at 16000g for 20 minutes. Discarding the supernatant, adding 100 μ l of mitochondrial protein extract (0.5-1.5 wt% of ethylphenylpolyethylene glycol, 0.5-1.5 wt% of polyethylene glycol octylphenyl ether, 0.1-0.5 wt% of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, 100-250mM sodium chloride, 2-10mM EDTA 20-70mM Tris, and adjusting pH to 6.5-8 with hydrochloric acid) into the precipitate to obtain the mitochondrial natural protein extract. Compared with the commercial kit of the conventional method, fig. 9 shows the results of the electrophoretic analysis, 1 shows the method of this embodiment, 2 shows the picea bottom mitochondrial protein extraction kit (cat No. C3601), and 3 shows the Thermo Scientific animal cell mitochondrial protein extraction kit (cat No. 89874), and the method of this embodiment extracts mitochondrial protein with higher concentration than the conventional extraction method, and saves the operation time.
It should be noted that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art should also make changes, modifications, additions or substitutions within the spirit and scope of the present invention.
Any equivalent embodiments that may be changed or modified into equivalent variations by those skilled in the art can be applied to other fields without departing from the technical scope of the present invention, and any simple modification, equivalent variation and modification made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical scope of the present invention.
Claims (7)
1. An automatic extraction method of cell components is characterized in that an instrument used in the method comprises an automatic extraction device of the cell components, and reagents used in the method comprise a sensitizer and a series of cell protein extraction solutions;
the automatic cell component extraction device includes: the device comprises a liquid transferring system, a filtering system, a cooling system and an automatic control system, wherein the cooling system provides low temperature of 0-4 ℃ for the liquid transferring system and/or the filtering system, and the automatic control system is respectively connected with the liquid transferring system, the filtering system and the cooling system; the filtration system includes: the cell membrane-sensitized cell culture device comprises a filter tube placing box (7), a ventilation and pressure supply system (6) and a filter tube (9), wherein the filter tube (9) comprises a filter column (9-3) and a collecting tube (9-1), the filter column (9-3) comprises a filter element (9-2), the filter element (9-2) breaks cells sensitized by cell membranes in the process of compressed gas pressure filtration and filters nucleic acid DNA, and the obtained filtrate is other cell components without DNA; the pipetting system comprises: a pipette tip placing box (1), a sample adding groove (8), a pipette (2) and a pipette tip; the pipettors (2) are arranged in rows and are arranged in parallel with the sample adding grooves, the sample adding grooves are sharp-bottom round hole groups communicated in rows, and the number and the positions of the sample adding grooves correspond to the pipette suction head placing box holes; the ventilation pressure supply system (6) comprises a fence (6-1), a compressed gas cylinder (6-4), a ventilation pipeline (6-5) and an air nozzle (6-7); the compressed gas cylinder is connected with the air nozzle through an air duct, a main valve (6-6) is arranged between the compressed gas cylinder and the air duct, the air duct is connected with the air nozzle through threads, a branch valve (6-3) is arranged between the air duct and the air nozzle, and a rubber gasket (6-2) is arranged at the contact part of the air nozzle (6-7) and the filter tube;
the sensitizer comprises polyethylene glycol octyl phenyl ether, ethyl phenyl polyethylene glycol and a buffer salt solution;
the series of cell component extracts comprises: polyethylene glycol octyl phenyl ether, ethyl phenyl polyethylene glycol, polyoxyethylene sorbitan monolaurate, 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, and a buffer salt solution;
the method comprises the following steps:
(1) placing the suction head into a suction head placing box of a pipette, placing a predetermined number of filter tubes into the filter tube placing box, and finally mixing a certain amount of cell sample and a sensitizer and adding the mixture into a sample adding groove for incubation;
(2) starting an automatic extraction program, after incubation for 5-10min, moving a pipettor to the position above a pipette tip placing box, and downwards moving and inserting a pipette tip; moving the pipettor to the upper part of the sample adding groove, and downwards moving to suck the sample mixed liquid; moving the pipettor to the upper part of the filter tube placing box, and moving downwards to add the sample into the filter tube; moving the pipettor back to the home position;
(3) the ventilation pressure supply system moves downwards, the air nozzle is inserted into the filter tube, the upper edge of the filter tube is tightly pressed and sealed with the rubber gasket, the air nozzle sprays pressurized gas, so that cell suspension treated by the sensitizing agent rapidly passes through the filter element, cells are crushed under the action of shearing force and inertial impact, and the ventilation pressure supply system moves upwards to the original position; taking out the filter tube, and collecting the filtrate in the tube, namely other cell components without DNA;
(4) if the total cell protein or the protein with a certain subcellular structure specially distributed is required to be collected, the product obtained in the step (3) can be centrifuged to obtain a subcellular structure precipitate, then the serial cell component extract is added, and the product obtained after all insoluble substances are dissolved is the specific protein extract contained in a certain subcellular structure.
2. The method according to claim 1, wherein the filter element is a microporous structure made of hydrophobic polyolefin polymer material.
3. The method according to claim 2, wherein the polymer material comprises polyethylene, polypropylene, or polytetrafluoroethylene.
4. The method as claimed in claim 2, wherein the filter element is made of polymer material into particles with a diameter of 10-150 μm, and is sintered into a microporous structure by pressing, wherein the diameter of the pores is 20-60 μm, and the thickness is 1mm-10 mm.
5. The method for automatically extracting cell components according to any one of claims 1, 2, 3 and 4, wherein the gas used by the aeration pressure supply system is nitrogen, and the cooling system can be a water-cooled heat exchange pipe connected with ice water or a compressor cooling system.
6. The method of claim 1, wherein the cell sensitizer comprises 0.10 wt% ~ 2.00.00 wt% of polyethylene glycol octylphenyl ether, 0.10 wt% ~ 2.50.50 wt% of ethylphenylpolyethylene glycol, and a buffered saline solution, wherein the buffered saline solution comprises 10-250mM sodium chloride, 1-20mM magnesium chloride, 20-70mM Tris-HCl buffered saline solution, pH 6.5-8.0, or 0.1- ~ 3 times PBS phosphate buffered saline solution, and pH 6.5-8.0.
7. The method as claimed in claim 1, wherein the cell fraction extraction liquid comprises 0.30 wt.% ~ 2.20.20 wt.% of polyethylene glycol octylphenyl ether, 0.10 wt.% ~ 4.00.00 wt.% of polyoxyethylene sorbitan monolaurate, 0.20 wt.% ~ 2.10.10 wt.% of ethylphenylpolyethylene glycol, 0.10 wt.% ~ 3.00.00 wt.% of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, and the buffer system comprises 100 mM sodium chloride, 20-70mM Tris-HCl buffer solution, pH 6.5-8.0 or 0.1- ~ 3-fold PBS buffer solution, and pH 6.5-8.0.
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