CN107557352A - The method of ultrasonic solid phase smudge cells and application - Google Patents
The method of ultrasonic solid phase smudge cells and application Download PDFInfo
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- CN107557352A CN107557352A CN201710830783.XA CN201710830783A CN107557352A CN 107557352 A CN107557352 A CN 107557352A CN 201710830783 A CN201710830783 A CN 201710830783A CN 107557352 A CN107557352 A CN 107557352A
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Abstract
The invention discloses a kind of method of ultrasonic solid phase smudge cells and application.The present invention is by cell sample after pre-treatment, accurate setting ultrasonic power and sonication treatment time, pretreated cell sample is carried out into ultrasonication.The present invention provides the technical scheme using ultrasonic solid phase crush method smudge cells first, the characteristics of for cell, accurate ultrasound condition is set, especially accurately determine ultrasonic power and the time being ultrasonically treated, overcome that cost during clasmatosis is high, easily causes being ramping up of temperature, the defects of product easily inactivates, the effect highly significant of clasmatosis.The inventive method is directed to clasmatosis problem, solves the problems, such as the clasmatosis of all kinds of strains, tissue, biological specimen well, in particular for histocyte and bacterial cell through broken extraction protein or DNA aspects.
Description
Technical field
The present invention relates to ultrasonic solid phase crushing technology field, more particularly, to a kind of side of ultrasonic solid phase smudge cells
Method and application.
Background technology
Smudge cells, various materials therein are extracted, analyzing the development of the structure and inhereditary material of cell to the mankind has
Important meaning.The difficulty or ease of clasmatosis are decided by its type.Existing clasmatosis includes chemical disruption methods, Mechanical Crushing
Method and physical disruption methods.Chemical disruption methods have osmotic shock method, surface active agent solubilization method or organic solvent dissolution method,
These chemical methods are gentleer, but product will not also produce irreversible denaturation after cytoclasis, and scale is easily amplified.It is such as blood red
The destruction of cell typically uses osmotic shock method, destroys cell by osmotic pressure, effect is gentleer, and cost is relatively low；Large intestine bar
Bacterium typically uses solubilized method, acts on Escherichia coli by surfactant cholate, effect is gentleer, moderate cost；Yeast
Cell typically uses organic solvent dissolution method, dissolves cell membrane with organic solvent toluene and is allowed to unstability, and effect is more moderate, into
This is relatively low.These methods respectively have advantage and disadvantage, although such as enzyme digestion mild condition, have selectivity, it is expensive.Have
Method destroy cell membrane but also destroy product.
Mechanical Method includes homogenate method (pass or piece type), polishing, supercritical ultrasonics technology, bead mill method, passes through high shearing
Power destroys cell.Animal tissue and zooblast split typically using piece type homogenate method, by cell by agitator it is broken, effect and into
This is moderate；The processing on a large scale of cell suspending liquid is existing typically using pass homogenate method, the aperture that must pass through cell, makes thin
Born of the same parents are crushed by shearing force；The extensive processing of plant cell typically uses bead mill method, by cell by bead or
Iron shot is smashed to pieces.Existing ultrasonic applications break cell in the small-scale processing of cell suspending liquid with the cavitation of ultrasonic wave
It is broken.The generation of heat is the defects of Mechanical Method can not overcome.
Physical disruption methods mainly make the broken method of histocyte by various physical factors.It is main to include freeze-thaw repeatedly
Method, anxious hot quenching method, ultrasonication etc..Wherein ultrasonication is the ultrasonication cell suspension with certain power, is made
Cell drastically shakes rupture.Ultrasonic wave is a kind of elastic mechanical ripple in material medium, and it is both a kind of wave, is one again
Kind form of energy.Effect of the ultrasound to cell mainly has fuel factor, cavitation effect and mechanical effect.Fuel factor is when ultrasound is being situated between
When being propagated in matter, frictional force hinders the molecular vibration as caused by ultrasound, portion of energy is converted into local high fever.Cavitation effect
It is under ultrasonic irradiation, vacuole is formed in organism, produces mechanical shear with vacuole vibrations and its fierce implosion and hob
Power and upheaval.In addition, producing TRANSIENT HIGH TEMPERATURE high pressure during cavitation follicular rupture, steam pyrolysis can be made from generation .OH free radicals and .H
Atom, the redox reaction as caused by .OH free radicals and .H atoms can cause polymer degraded, enzyme inactivation, lipid peroxidation
And cell killing.Mechanical effect is the primary effect of ultrasound, and ultrasonic wave medium particle in communication process alternately compresses and stretched
Open and constitute pressure change, cause eucaryotic cell structure to be damaged.The frequency and intensity of the power and ultrasound of damaging action are closely related.It is super
In sonication, acoustic energy transmission, radiating is had any problem, it is necessary to take targetedly cooling measure.Cavitation is cytoclasis
Immediate cause, at the same can produce active oxygen and chemical free-radical group easily make products inert, so it has impact on it large-scale
Industrial application.Due to a variety of limitations, ultrasonic fragmentation not can apply to the cell and core sensitive to ultrasonic wave at present
Acid.
The applicant discloses a kind of ultrasonic wave microorganism in the Chinese patent application of Application No. 2014105033514
Process for dispersing and device.Ultrasonic wave is directly acted on to the outer of the container that holds microorganism to be disperseed after ultrasonic transducer
Wall, ultrasonic wave is directly delivered to by wall and treat in dispersed sample, it is not necessary to by liquid medium, then utilize ultrasonic cavitation
Effect, reach and microbic mass is subjected to scattered and de-agglomerated effect, so as to reach scattered purpose.So in dispersion process both
Can seal it is scattered, while it also avoid wall surface hang attached liquid body, influence subsequent operation.But this method is during application
Limitation is equally existed, is only limited to be applied to the strain processing that more obstinate difficulty is disperseed, is such as applied to tulase bacterium
Break up.Have no that ultrasonication method is successfully applied to the research breakthrough of the broken aspect of strain at present, that is, it is thin to be applied to broken sample
The research of cell wall, more have no and methods described is applied to the broken aspect of histocyte.And after successful fracture sample cell membrane
Conveniently follow-up further extraction protein, DNA etc. if the method for simple and easy ultrasonic solid phase smudge cells can be used to break
Broken strain or histocyte, will be that the scientific research activities such as detection experiment bring breakthrough sexual revolution and establish essence technology base
Plinth.
The content of the invention
The technical problem to be solved in the present invention is the crushing technology deficiency for existing cell, there is provided a kind of ultrasonic solid phase is broken
The method of chopping fine born of the same parents.
Another technical problems to be solved of the present invention are to provide the application of the ultrasonic solid phase smudge cells method.
The purpose of the present invention is achieved by the following technical programs：
A kind of method of ultrasonic solid phase smudge cells is provided, comprised the following steps：
S1. cell sample is preprocessed, obtains pretreatment sample；
S2. ultrasonic wave is directly acted on after ultrasonic transducer and holds the cell sample through step S1 pretreatments
Container outer wall, ultrasonic power and ultrasonic time are set, the cell sample pre-processed through step S1 is ultrasonically treated, at ultrasound
Centrifuging and taking supernatant after reason；
Wherein, the ultrasonic power being ultrasonically treated described in step S2 is 40W~180W；Ultrasonic time is 10~40min.
Preferably, the ultrasonic power being ultrasonically treated described in step S2 is 80W~180W.
Preferably, the ultrasonic power being ultrasonically treated described in step S2 is 160W~180W.
Preferably, the ultrasonic power being ultrasonically treated described in step S2 is 180W.
Invention also provides the application of the method for the ultrasonic solid phase smudge cells, can be advantageously applied to crush
In terms of histocyte or bacterial cell, in terms of especially extracting cell protein or DNA through clasmatosis.
Centrifuging and taking supernatant after step S2 ultrasounds of the present invention, surveys OD respectively260And protein concentration, statistical data analysis.
Methods described is HeLa cell, red blood cell or animal liver cell in histocyte；The bacterium is aspergillus niger, large intestine
When bacillus or saccharomycete are application, effect is especially notable.
Cell sample described in the inventive method application can be the groups such as HeLa cell, red blood cell, animal liver cell
Knit cell；For histocytes such as the HeLa cell, red blood cell, animal liver cells, the ultrasound that is ultrasonically treated described in step S2
Power is preferably 80W~180W, and ultrasonic time is preferably 10~40min.It is highly preferred that ultrasonic power described in step S2 is 120W
~180W, ultrasonic time are 30~40min.Most preferably, ultrasonic power described in step S2 is 160W~180W, and ultrasonic time is
30~40min.
Cell sample described in the inventive method application can be the bacteriums such as aspergillus niger, Escherichia coli or saccharomycete
Cell.For the cell of the bacteriums such as the aspergillus niger, Escherichia coli or saccharomycete, the ultrasonic power that is ultrasonically treated described in step S2
Preferably 80W~180W, ultrasonic time are preferably 10~40min.It is highly preferred that ultrasonic power described in step S2 80W~
Under the conditions of 180W, ultrasonic time is preferably 30~40min；Ultrasonic power described in step S2 is under the conditions of 160W~180W, ultrasound
Time is preferably 10~40min.
Cell sample in the inventive method application when processing is HeLa cell, and the pretreatment of its cell sample is preferred
Step is handled in accordance with the following methods：Pretreatment includes digesting, neutralize, be collected by centrifugation and being resuspended processing step；The digestion
Be toward HeLa cell in add 0.25% (m/v) pancreatin, mix, be placed in 37 DEG C of incubators 5~6min of digestion；The neutralization is to see
Examine after HeLa cell digested completely, add DMEM (L) nutrient solution and terminate digestion, piping and druming repeatedly mixes；Described be collected by centrifugation be
After trim under the conditions of 200 × g centrifugal treating 3min, collect centrifugal treating after neutralizer；The resuspension is by new DMEM
Nutrient solution adds the neutralizer being collected by centrifugation, and piping and druming repeatedly makes cell fully mix.
Cell sample described in the inventive method application is red blood cell, and the ultrasonic power of the supersound process is preferably
160W~200W；Ultrasonic time is 10~40min.The preprocess method of its cell sample is to take anticoagulation to add equivalent blood physiology salt
Water, mix, 2000r/min centrifugation 5min, remove supernatant；With without Ca2+、Mg2+Hanks liquid is by red blood cell washing 3 times, first 2 times
2000r/min, time are respectively 5min, last 2000r/min, time 10min；Supernatant is removed, packed red cells is made into institute
Concentration is needed to produce.
Cell sample described in the inventive method application is animal liver cell, and the preprocess method of its cell sample is
Animal's liver sample is taken, the hepatic tissue of elution is shredded and is put into homogenizer, cold TE buffer solutions is added, is fully ground,
Homogenate is made；Homogenate is taken to add equivalent PBS, supernatant is abandoned in centrifugation, and addition PBS, which redissolves, to be produced.
Cell sample described in the inventive method application is aspergillus niger, and the preprocess method of its cell sample is will be black
Aspergillus point, which is planted, to be cultivated in sterilizing flat board, being inverted in 25~28 DEG C of incubators；By the streak inoculation of mould pure culture in inclined-plane, training
Support 5~14d；Collect culture to be dissolved in PBS, centrifugation is abandoned after supernatant to be redissolved with PBS and produced.
Cell sample described in the inventive method application is saccharomycete, and the preprocess method of its cell sample is configuration
Liquid YPD medium, autoclave sterilization are followed by Yeasts, carry out shaking bacterium culture at 30 DEG C, will be abandoned after medium centrifugal
Supernatant, PBS is added, piping and druming repeatedly makes cell fully mix, and is counted under microscope, and stoste dilution debita spissitudo produces.
Cell sample described in the inventive method application is Escherichia coli, the preprocess method reference of its cell sample
Existing conventional techniques.
Beneficial effects of the present invention：
The art realizes the method smudge cells crushed using solid phase not successfully for a long time, and the present invention uses first
Ultrasonic solid phase crush method separates and collects different cells, the characteristics of for different cells, sets accurate ultrasound condition, especially really
The time determined the ultrasonic power of science and be ultrasonically treated, ensure the effect of the clasmatosis of highly significant, the inventive method is not only
The scattered of all kinds of strains can be advantageously applied to, strain, tissue, biological specimen can also be directed to and realize clasmatosis, especially
It is subsequently to be ground for extraction protein, DNA etc. through broken extraction protein or DNA aspects for histocyte and bacterial cell
Study carefully and lay solid technical foundation.
It is long-term to solve this area by determining accurate ultrasonic power and time-critical condition simple and easyly by the present invention
Existing technical barrier, not only ensure that clasmatosis effect, and after the processing from the point of view of mrna concentration and protein concentration data,
Targetedly crush with the technique effect especially with high efficiency extraction protein and DNA.Illustrate to pass through using the inventive method
Science controls to adjust the effect that crucial technological parameter can reaches clasmatosis.
On the basis of design philosophy of the present invention, the present invention further optimizes the pretreatment operation step and parameter of cell,
Laid a good foundation for follow-up ultrasonication.
Brief description of the drawings
Fig. 1 is that the inventive method crushes the DNA concentration situation after HeLa cell.
Fig. 2 is that the inventive method crushes the protein concentration situation after HeLa cell.
Fig. 3 is the DNA concentration situation after the inventive method broken red blood cell.
Fig. 4 is the protein concentration situation after the inventive method broken red blood cell.
Fig. 5 is that the inventive method crushes the DNA concentration situation after mouse liver cell.
Fig. 6 is that the inventive method crushes the protein concentration situation after mouse liver cell.
Fig. 7 is that the inventive method crushes the DNA concentration situation after Bacillus coli cells.
Fig. 8 is that the inventive method crushes the protein concentration situation after Bacillus coli cells.
Fig. 9 is that the inventive method crushes the DNA concentration situation after aspergillus niger cell.
Figure 10 is that the inventive method crushes the protein concentration situation after aspergillus niger cell.
Figure 11 is the DNA concentration situation after the inventive method Breaking Yeast bacterium cell.
Figure 12 is the protein concentration situation after the inventive method Breaking Yeast bacterium cell.
Embodiment
The present invention is further illustrated with reference to specific embodiment.The ore source that following embodiments illustrate is given for example only
Property explanation, it is impossible to be interpreted as limitation of the present invention.For convenience of explanation, the embodiments of the invention provide giving ore deposit source, but simultaneously
Therefore the scope of the invention is not limited.Unless stated otherwise, the raw material used in following embodiments is this area conventional market channel
The raw material of acquisition, unless stated otherwise, the method and apparatus used in following embodiments be method commonly used in the art and
Equipment.
The ultrasonication HeLa cell of embodiment 1
S1. the pretreatment of cell sample：
Digestion：HeLa cell is placed in blake bottle, 5mL pipettes are inserted in electric rifle, alcolhol burner overdoes calcination, draws
2mL0.25% pancreatin is added in blake bottle, is mixed, and is put in 5~6min of digestion in 37 DEG C of incubators.
Neutralize：Blake bottle is taken out from incubator, whether micro- Microscopic observation cell is digested completely, after 5mL sterilizings
Pipette adds 3mLDMEM nutrient solutions and terminates digestion, and piping and druming repeatedly mixes, and suctions out neutralizer and is transferred in 50mL centrifuge tubes.
Cell is collected by centrifugation：200 × g, 3min are centrifuged after trim,
Cell is resuspended：New DMEM nutrient solutions are added in new blake bottle, 2mL/ bottles, suction out the neutralizer after centrifugation, add
Enter in the new blake bottle for having added new DMEM nutrient solutions, electricity consumption rifle is blown and beaten repeatedly makes cell fully mix, and is counted under microscope, stoste
Debita spissitudo is diluted, adds 3mL to PC to manage.
S2. by ultrasonic wave by ultrasonic transducer (with reference to the equipment disclosed in Application No. 2014105033514, under
It is same to state embodiment) after directly act on hold through step S1 pretreatment cell sample container (PC pipes) outer wall, set respectively
Ultrasonic power and ultrasonic time, are ultrasonically treated, and centrifugal treating 3min takes supernatant under 200 × g after ultrasound, surveys A260 respectively
And protein concentration, shown in experimental result as Fig. 1 and Fig. 2.
The ultrasonication red blood cell of embodiment 2
S1. the pretreatment of cell sample：Take anticoagulation to add equivalent blood physiology salt solution, mix, 2000r/min centrifugation 5min,
Remove supernatant；With without Ca2+、Mg2+Hanks liquid by red blood cell washing 3 times, preceding 2 2000r/min, 5min, last 2000r/min,
10min, supernatant is removed, packed red cells is made into required concentration, add 3mL to PC to manage.
S2. ultrasonic wave is directly acted on after ultrasonic transducer and holds the cell sample through step S1 pretreatments
Container (PC pipes) outer wall, set different ultrasonic powers and ultrasonic time progress ultrasonic, centrifuging and taking supernatant after ultrasound, survey egg respectively
White concentration and calculating percentage of damage.Shown in experimental result as Fig. 3 and Fig. 4.
The ultrasonication animal liver cell of embodiment 3
S1. the pretreatment of cell sample：Mouse liver is taken, the hepatic tissue of elution is shredded and is put into homogenizer, is being added
TE buffer solutions cold 5mL, are fully ground, homogenate are made.5mL homogenates are taken to add equivalent PBS, centrifugation is abandoned supernatant, added
Enter PBS redissolution, take 3mL to PC to manage.
S2. ultrasonic wave is directly acted on after ultrasonic transducer and holds the cell sample through step S1 pretreatments
Container (PC pipes) outer wall, set different ultrasonic powers and ultrasonic time progress ultrasonic, centrifuging and taking supernatant after ultrasound, survey egg respectively
White concentration and calculating percentage of damage.Shown in experimental result as Fig. 5 and Fig. 6.
The cell of the ultrasonication Escherichia coli of embodiment 4
S1. the pretreatment of cell sample：
S2. ultrasonic wave is directly acted on after ultrasonic transducer and holds the cell sample through step S1 pretreatments
Container (PC pipes) outer wall, set different ultrasonic powers and ultrasonic time progress ultrasonic, centrifuging and taking supernatant after ultrasound, survey egg respectively
White concentration and calculating percentage of damage.Shown in experimental result as Fig. 7 and Fig. 8.
The cell of the ultrasonication aspergillus niger of embodiment 5
S1. the pretreatment of cell sample：Czapek'S medium 50g is weighed, adds distilled water or deionized water 1L, agitating and heating
Boil to being completely dissolved, dispense triangular flask, 121 DEG C of autoclaving 20min, wait that being cooled to 55~60 DEG C pours into sterilizing flat board；Will be black
Aspergillus point, which is planted, to be cultivated on flat board, being inverted in 25~28 DEG C of incubators.Observe on inclined-plane：By mould pure culture streak inoculation in oblique
Face, 5~14d is cultivated, observe colonial morphology, while strain tube can also be put under microscope and directly observe spore with low power lens
Form and arrangement.Collect culture to be dissolved in PBS, centrifugation is redissolved after abandoning supernatant with PBS, adds 3mL to PC to manage.
S2. ultrasonic wave is directly acted on after ultrasonic transducer and holds the cell sample through step S1 pretreatments
Container (PC pipes) outer wall, set different ultrasonic powers and ultrasonic time progress ultrasonic, centrifuging and taking supernatant after ultrasound, survey egg respectively
White concentration and calculating percentage of damage.Shown in experimental result as Fig. 9 and Figure 10.
The cell of the ultrasonication saccharomycete of embodiment 6
S1. the pretreatment of cell sample：Liquid YPD medium is configured, autoclave sterilization is followed by Yeasts, at 30 DEG C
Under shake bacterium culture.Cell is collected by centrifugation and cell is resuspended：Supernatant is abandoned after centrifugation, adds PBS, piping and druming repeatedly makes cell abundant
Mix, counted under microscope, stoste dilution debita spissitudo, add 3mL to PC to manage.
S2. ultrasonic wave is directly acted on after ultrasonic transducer and holds the cell sample through step S1 pretreatments
Container (PC pipes) outer wall, set different ultrasonic powers and ultrasonic time progress ultrasonic, centrifuging and taking supernatant after ultrasound, survey egg respectively
White concentration and calculating percentage of damage.Shown in experimental result as Figure 11 and Figure 12.
Claims (10)
- A kind of 1. method of ultrasonic solid phase smudge cells, it is characterised in that comprise the following steps：S1. cell sample is preprocessed, obtains pretreatment sample；S2., ultrasonic wave is directly acted on to the container for holding the cell sample through step S1 pretreatments after ultrasonic transducer Outer wall, ultrasonic power and ultrasonic time are set, the cell sample pre-processed through step S1 are ultrasonically treated, after supersound process Centrifuging and taking supernatant；The ultrasonic power being ultrasonically treated described in wherein step S2 is 40W~180W；Ultrasonic time is 10~40min.
- 2. the method for ultrasonic solid phase smudge cells according to claim 1, it is characterised in that be ultrasonically treated described in step S2 Ultrasonic power is 80W~180W.
- 3. the method for ultrasonic solid phase smudge cells according to claim 1, it is characterised in that be ultrasonically treated described in step S2 Ultrasonic power is 160W~180W.
- 4. the method for ultrasonic solid phase smudge cells according to claim 1, it is characterised in that be ultrasonically treated described in step S2 Ultrasonic power is 180W.
- 5. the application of the method for any one of Claims 1-4 ultrasonic solid phase smudge cells, it is characterised in that applied to broken Broken histocyte or bacterial cell.
- 6. application according to claim 5, it is characterised in that the histocyte is HeLa cell, red blood cell or animal Liver cell；The bacterium is aspergillus niger, Escherichia coli or saccharomycete.
- 7. application according to claim 6, it is characterised in that the cell sample is histocyte, is surpassed described in step S2 The ultrasonic power of sonication is 80W~180W, and ultrasonic time is 10~40min；Ultrasonic power described in step S2 is preferably 120W ~180W, ultrasonic time are preferably 30~40min；Ultrasonic power described in step S2 is more preferably 160W~180W, ultrasonic time More preferably 30~40min.
- 8. application according to claim 7, it is characterised in that the cell sample is HeLa cell, its cell sample Pretreatment includes digesting, neutralize, be collected by centrifugation and being resuspended processing step；It is described digestion be toward HeLa cell in add 0.25% （m/v）Pancreatin, mix, be placed in 5~6min of digestion in 37 DEG C of incubators；It is described neutralization be observation HeLa cell digested completely after, Add DMEM nutrient solutions and terminate digestion, piping and druming repeatedly mixes；Described be collected by centrifugation is under the conditions of 200 × g at centrifugation after trim 3min is managed, collects the neutralizer after centrifugal treating；The resuspension is that new DMEM nutrient solutions are added to the neutralizer being collected by centrifugation, Piping and druming makes cell fully mix repeatedly；Or the cell sample is red blood cell, the preprocess method of its cell sample is to take anticoagulation to add equivalent blood physiology salt Water, mix, 2000r/min centrifugation 5min, remove supernatant；With without Ca2+、Mg2+Hanks liquid is by red blood cell washing 3 times, first 2 times 2000r/min, time are respectively 5min, last 2000r/min, time 10min；Supernatant is removed, packed red cells is made into institute Concentration is needed to produce；Or the cell sample is animal liver cell, the preprocess method of its cell sample is to take animal's liver sample, will be washed De- hepatic tissue, which shreds, to be put into homogenizer, is added cold TE buffer solutions, is fully ground, homogenate is made；Take homogenate Equivalent PBS is added, supernatant is abandoned in centrifugation, and addition PBS, which redissolves, to be produced.
- 9. application according to claim 7, it is characterised in that the cell sample be bacterium cell sample, step S2 The ultrasonic power of the supersound process is 80W~180W；Ultrasonic time is 10~40min；Ultrasonic power described in preferred steps S2 Under the conditions of 80W~180W, ultrasonic time is 30~40min；Ultrasonic power is in 160W~180W bars more preferably described in step S2 Under part, ultrasonic time is 10~40min.
- 10. application according to claim 9, it is characterised in that the cell sample is aspergillus niger, its cell sample it is pre- Processing method is to plant aspergillus niger point to cultivate in sterilizing flat board, being inverted in 25~28 DEG C of incubators；Mould pure culture is drawn Line is inoculated in inclined-plane, cultivates 5~14d；Collect culture to be dissolved in PBS, centrifugation is abandoned after supernatant to be redissolved with PBS and produced；Or the cell sample is saccharomycete, the preprocess method of its cell sample is to configure liquid YPD medium, high temperature Autoclaving is followed by Yeasts, carries out shaking bacterium culture at 30 DEG C, supernatant will be abandoned after medium centrifugal, PBS is added, blows repeatedly Beating makes cell fully mix, and is counted under microscope, and stoste dilution debita spissitudo produces.
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| CN112159804A (en) * | 2020-09-16 | 2021-01-01 | 湖南省动物疫病预防控制中心 | Tick nucleic acid extraction method |
| CN114292805A (en) * | 2022-01-10 | 2022-04-08 | 中国原子能科学研究院 | Method for fully extracting total protein of adherent cells |
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| CN114292805A (en) * | 2022-01-10 | 2022-04-08 | 中国原子能科学研究院 | Method for fully extracting total protein of adherent cells |
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