CN107522771A - Tapetal cell total protein extraction method - Google Patents
Tapetal cell total protein extraction method Download PDFInfo
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- CN107522771A CN107522771A CN201710976855.1A CN201710976855A CN107522771A CN 107522771 A CN107522771 A CN 107522771A CN 201710976855 A CN201710976855 A CN 201710976855A CN 107522771 A CN107522771 A CN 107522771A
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- total protein
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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
- C07K1/30—Extraction; Separation; Purification by precipitation
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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
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention provides a kind of method for separating, extracting tapetal cell total protein, content includes：Total protein extraction formula of liquid, tapetal cell is collected and its total protein extraction and collection.Total protein extracting solution main component：Tris HCl, SDS, β mercaptoethanol, TritonX 100, Borax, EDTA, Vc, PVPP.Total protein of cell extracts flow：Collect tapetal cell-grinding cell -110 DEG C, 30min；80 DEG C, albumen is collected in 2h-separation total protein solution-low-temperature precipitation albumen-washing.This method has the advantages that extraction efficiency is high, quality is good, reproducible, simple to operate, the used time is short, and good technology platform is provided for the accurate proteomics research of target plant cells.
Description
Technical field
The invention belongs to biology techniques research field, is a kind of more particularly, to protein stripping technique research field
From target cell easily and fast, accurate separation and Extraction gross protein technology category.
Background technology
The large biological molecule of targeting separation higher plant body aim cell or tissue carries out its accurate molecular biology research,
It is the great difficult problem for perplexing scientists in current plant science area research.And in the technology of biological study at present, carry out target
Completed mostly using paraffin section auxiliary laser micro-dissections and antigen retrieval method to cell separation large biological molecule technology.Should
Technology application antigen retrieval principle, skill is operated by professional tabletting technology and specialized cells tissue cutting device and antigen retrieval
Art reaches the protein of parting tissue or cell and other biological macromolecular purpose.The technology is the last century 80's
End is invented by foreign countries, is generally used in medical pathologies molecular biology afterwards.But the requirement limitation due to the technology in itself
It is widely popularized use in field of biology.Specific defect shows that one side paraffin section technology belongs to the aobvious of specialty
Micro- tableting operation technology is, it is necessary to which well-trained professional and technical personnel completes, and paraffin wax flaking operating process is not only tediously long but also very
It is complicated, cumbersome.Another aspect target cell obtains will could also complete by the laser microprobe dating instrument cutting operation of specialty.Again
Person, the heater that uses is varied in antigen retrieval operation, such as water-bath, micro-wave oven, pressure cooker, normal domestic use pot, disappears
Xie Yi etc., it result in reparation result and be difficult to be precisely controlled.Therefore, the technology used at present had both limited the accurate life of target cell
The development of thing research, the large biological molecule result of study stability that also result in acquisition is precisely controlled, such as Chinese patent《One
Kind method for extracting protein from paraffin-embedded tissue》（The patent No.:200510120873））,《It is a kind of from formalin-fixed tissue or first
Aldehyde, which is fixed, extracts total protein method in paraffin-embedded tissue》（The patent No.：201010286673.X）Etc..
Therefore, up to the present there has been no a kind of convenience, simple and direct suitable target cell precisely to separate the complete of large biological molecule
Whole isolation technics.
The content of the invention
It is an object of the invention to provide a kind of method for precisely separating and collecting gross protein suitable for target cell.
Technical scheme is as follows：Tapetal cell total protein extraction method, the base program of this method include with
Lower step：
1st, fixed tapetal cell is collected in mill；
2nd, add up protein extract and quickly grind tapetal cell, cell slurry moves into centrifuge tube afterwards, seals the pipe of centrifuge tube
Mouthful；
3rd, centrifuge tube is placed in metal bath, in 110 DEG C of temperature, keeps 30min；Then 80 DEG C are down to, keeps 2h, period 20min
Mix 1 time；
4th, in 4 DEG C, 16000 × g, 15 min, centrifuge and repair total protein solution；
5th, repair total protein supernatant to move into new centrifuge tube, add 5 times of volume of protein precipitation reagents, -20 DEG C of overnight precipitation albumen；
6th, successively add the different albumen washing reagent of 3 times of volumes, gently mix, stand 10 min（In ice bath）, 4 DEG C, 16000 ×
G, 5 min, washing, separation total protein；
7th, total protein is dried.
Points for attention in implementation process of the present invention：
（1）The tapetal cell collection method need it is important to note that be cell set time length with albumen reparation with extraction extremely
Close important.It is 3.5-4h that tapetal cell of the present invention, which fixes Best Times,.
（2）The tapetal cell fixes agent prescription：2% paraformaldehyde, 10% polyethylene glycol,
0.1M phosphate buffers, PH7.2（Adjusted with glacial acetic acid）.
（3）The total protein repairs extract solution constituent：Tris-HCl, SDS, beta -mercaptoethanol, Borax,
TritonX-100、EDTA、Vc、PVPP.Suitable concentration range is：Tris-HCl 65-200mM, SDS 2-4%, β-sulfydryl second
Alcohol 5-25%, TritonX-100 1%, Borax40-60mM, EDTA 60-100mM, Vc50mM, PVPP1%, buffer system
PH2-10, it is best suitable for PH8.0.Solution formula is extracted in total protein reparation of the present invention：95 or 200mMTris-HCl, 3.5%SDS,
25% beta -mercaptoethanol, 50mMBorax, 1%TritonX-100,100mMEDTA, 50mMVc, 1%PVPP, PH8.0.
（4）Cell sample repairs extracting liquid volume ratio about 1 with total protein：3-10, preferable 1：10.
Cell lapping apparatus is preferably：Grind alms bowl, cell tissue mill.
（5）The heater used in total protein of cell restorative procedure is preferably：Metal bath.
（6）Total protein high temperature reparation operation stablizes 2 groups of condition of different temperatures combinations for high temperature hot repair is multiple with slightly high temperature incubation
Carry out.The multiple heating-up temperature of high temperature hot repair is preferably 95-120 DEG C, and optimal is preferably 110 DEG C；The high temperature hot repair multiple heat time is preferred
For 20-30min, preferably most preferably 30min.Slightly higher preferably 60 DEG C or 80 DEG C, preferably most preferably 80 DEG C of heated culture temperature of warm；Slightly higher temperature
It is preferably 2h to incubate stabilization time.It is especially noted that when high temperature reparation and the slightly temperature of high temperature incubation stabilization and heating
Between to albumen remediation efficiency even repair success or failure it is most important.After high temperature hot repair multiple junction beam, egg white repairing liquid has to nature drop
Warm to room temperature, otherwise effect is bad or does not reach albumen reparation purpose, and sample, when high temperature hot repair is multiple, the centrifuge tube mouth of pipe is engaged in
High heat-proof rubber belt sealing must be used, with the high pressure purpose that reaches a high temperature.
（7）Repairing protein solution centrifugation operation preferably 4 DEG C of low-temperature centrifugations, centrifugal force is preferably： 13000-16000
× g, preferably most preferably 16000 × g.Centrifugation time is preferably 15-20min, most preferably preferably 15 min.
（8）Precipitation of protein is preferably：Phenol ammonium sulfate precipitation method, ammonium sulfate methanol extraction method, TCA acetone precipitations are pure
Acetone precipitation and acetone methanol extraction method.The optimum protein precipitation method are ammonium sulfate methanol extraction method, can also be phenol ammonium sulfate
Method and TCA acetone methods.
（9）Successively albumen precipitation is fully washed after albumen precipitation with ice methanol and ice acetone, and ice methanol and ice acetone
To be handled before in -20 DEG C of cryogenic freezings.
（10）Total protein collection method is boulton process and natural drying at room temperature method, and 2 kinds of methods are collected into
Albumen powder does not influence final albumen quality.
The present invention has following remarkable result：
1st, method of the present invention and existing FFPE（Or combine laser microprobe dating isolation technics）+ albumen restorative procedure is all adopted
Total protein technology is extracted with antigen retrieval principle, but the present invention can just complete without specialized equipment or professional training personnel, and
And operation is simple for method, the used time is short.
2nd, the inventive method is by the separation and Extraction total protein of cell on the basis of fixing and precisely obtaining target cell.Cause
This, more advantage more valuable for the accurate proteomics research of target cell.
3rd, the inventive method step is easy to standardize, and as a result repeats and stability is good.
4th, the inventive method is embodied within the extraction of plant tapetum total protein of cell, also may extend to plant other groups
The total protein extraction of cell is knitted, convenience, simple and direct research way are provided for the accurate proteomics research of higher plant target cell
Footpath, application easy to spread.
, can be with further genralrlization to animal etc. if the 5, the inventive method slightly adjusts to fixative and albumen renovation agent
The target cellular protein matter separation and Extraction of its hetero-organization.
Embodiment
In order that technical scheme is clearer, is readily appreciated that, with reference to embodiments, the present invention is carried out
It is further described.Described specific embodiment is only to explain the present invention, the protection being not intended to limit the present invention
Scope.
Embodiment 1
1) the fixation tapetal cell about 30mg for collecting certain developmental stage is ground in alms bowl in ice；
2）Add 300ul total proteins to repair extract solution and quickly grind tapetal cell, cell slurry is moved into centrifuge tube, medical adhesive
Band seals up the centrifugation mouth of pipe；
3）Centrifuge tube is placed in metal bath, 110 DEG C, 30min；Room temperature is naturally cooling to, 80 DEG C, 20min is mixed 1 time during 2h；
4）Centrifugation, 4 DEG C, 16000 × g, 15 min, supernatant is moved into new centrifuge tube；
5）5 times of volume ice ammonium sulfate methanol saturated solutions, -20 DEG C of overnight precipitation albumen are slowly added dropwise in supernatant；
6）4 DEG C of albumen precipitation liquid, 16000 × g, 5 min, stays precipitation；
7）Add 3 times of volume ice methanol, gently mix, stand 10 min（In ice bath）；
8）4 DEG C, 16000 × g, 5 min, stay precipitation；
9）It is repeated 1 times 7）With 8）Step；
10）Add 3 times of volume ice acetone, gently mix, stand 10 min（In ice bath）,
11）4 DEG C, 16000 × g, 5 min, stay precipitation；
12）It is repeated 2 times 10）With 11）Step；
13）Centrifuge tube is inverted on filter paper in fume hood, spontaneously dries protein dry powder about 20-30min.
The total protein dry powder that separation obtains is dissolvable in water protein lysate（Such as laemmli sample buffer）It is direct afterwards
Studied for subsequent protein and proteomic experiments, or -80 DEG C of low temperature longer-terms store for future use.
14) the fresh flower pesticide total protein of contemporaneity is carried out with fixed flower pesticide by the total protein of the inventive method extraction
SDS-PAGE electrophoresis compares confirmation, and the two protein band does not have notable difference.
Embodiment 2
1) the pollen about 20mg after fixing is collected to grind in alms bowl in ice；
2）Add 200ul total proteins to repair extract solution and quickly grind pollen, pollen slurry is moved into centrifuge tube, and medical adhesive tape seals
Centrifuge the mouth of pipe；
Subsequent process steps press the 3 of above-described embodiment 1）-13）Step is implemented, you can obtains pollen total protein dry powder.
The fresh pollen total protein of contemporaneity is subjected to SDS- with fixed pollen by the total protein of the inventive method extraction
PAGE electrophoresis compares confirmation, and the two protein band is highly consistent.
Claims (10)
1. tapetal cell total protein extraction method, it is characterised in that this method includes following operating procedure：
（1）Fixed tapetal cell is collected in mill；
（2）Add up protein extract and quickly grind tapetal cell, cell slurry moves into centrifuge tube afterwards, seals the pipe of centrifuge tube
Mouthful；
（3）Centrifuge tube is placed in metal bath, 110 DEG C, keeps 30min；Then 80 DEG C are down to, keeps 2h, period 20min to mix 1
It is secondary；
（4）4 DEG C, 16000 × g, 15 min, centrifuge and repair total protein solution；
（5）Repair total protein supernatant to move into new centrifuge tube, add 5 times of volume of protein precipitation reagents, -20 DEG C of overnight precipitation albumen；
（6）Successively add the different albumen washing reagent of 3 times of volumes, gently mix, stand 10 min（In ice bath）, 4 DEG C, 16000
× g, 5 min, washing, separation total protein；
（7）Dry total protein.
2. method according to claim 1, it is characterised in that the step（1）Tapetal cell fixes collection method：Stripping
Room temperature is immersed in fixative from flower pesticide and fixes 3.5-4h, is extruded, is beaten tapetal cell and flower pesticide its hetero-organization with spillikin
Separation, then successively filter out tapetal cell, 1000 × g, room temperature centrifugation by 80 mesh, 200 mesh and 600 mesh cell sieves again
5min, collect tapetal cell.Tapetal cell fixative is：2% paraformaldehyde, 10% polyethylene glycol, 0.1M phosphate buffers,
PH7.2（PH value is adjusted with glacial acetic acid）.
3. method according to claim 1, it is characterised in that the step（2）Total protein of cell repair extract solution composition into
It is divided into：Tris-HCl, SDS, beta -mercaptoethanol, Borax, TritonX-100, EDTA, Vc, PVPP；Wherein each component concentration model
Enclose for：Tris-HCl 65-300mM, SDS 2-4%, beta -mercaptoethanol 5-25%, TritonX-100 1%, Borax40-
60mM, EDTA 60-100mM, Vc50mM, PVPP1%, Tris-HCl buffer solution ph 8.0.
4. method according to claim 1, it is characterised in that the step（3）Total protein of cell reparation needs to use heating
Device, heater are preferably：Metal bath or water-bath, most preferably preferably metal bath.
5. method according to claim 4, it is characterised in that total protein reparation operation is high temperature reparation and slightly high temperature incubation is steady
Fixed 2 kinds of heating conditions combination is carried out：The multiple heating-up temperature of high temperature hot repair is preferably 95-120 DEG C, and optimal is preferably 110 DEG C；High warm
It is preferably 20-30min to repair the heat time, most preferably preferably 30min；Preferably 60 DEG C or 80 DEG C of slightly higher temperature heated culture temperature, most preferably
It is preferred that 80 DEG C；Slightly high temperature incubation stabilization time is preferably 2h.
6. method according to claim 4, it is characterised in that：After high temperature reparation terminates, egg white repairing liquid must use nature
Egg white repairing liquid is down to room temperature by cool-down method, and otherwise effect is bad or does not reach albumen reparation purpose.
7. method according to claim 1, the step（4）Repair total protein separation and extracting method, it is characterised in that：Repair
Recoverin solution centrifugal lock out operation preferably 4 DEG C of low-temperature centrifugations, centrifugal force are preferably：13000-16000 × g, it is optimal preferred
16000 × g, centrifugation time are preferably 15 min.
8. method according to claim 1, it is characterised in that the step（5）It is described to be repaiied using precipitation of protein to collect
Multiple total protein, albumen precipitation method are preferably：It is phenol ammonium sulfate precipitation method, ammonium sulfate methanol extraction method, TCA acetone precipitations, pure
Acetone precipitation and acetone methanol extraction method, the optimum protein precipitation method are ammonium sulfate methanol method, can also be phenol ammonium sulfate and TCA
Acetone precipitation.
9. method according to claim 1, it is characterised in that the step（6）After ammonium sulfate methanol extraction method protein precipitation,
Albumen precipitation is fully successively washed with ice methanol and ice acetone, and ice methanol and ice acetone will be in -20 DEG C of cryogenic freezings before using
Processing.
10. method according to claim 1, it is characterised in that the step（7）Total protein collection method be seasoning,
The inventive method is preferably dried in vacuo instrument seasoning or natural drying at room temperature method.
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|---|---|---|---|
| CN201710976855.1A CN107522771A (en) | 2017-10-19 | 2017-10-19 | Tapetal cell total protein extraction method |
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| CN201710976855.1A CN107522771A (en) | 2017-10-19 | 2017-10-19 | Tapetal cell total protein extraction method |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1803828A (en) * | 2005-12-28 | 2006-07-19 | 南方医科大学 | Method for extracting protein from paraffin-embedded tissue |
| CN101475634A (en) * | 2009-02-05 | 2009-07-08 | 中国科学院植物研究所 | Extracting method for plant chloroplast total protein and special extract therefor |
| CN101696238A (en) * | 2009-10-27 | 2010-04-21 | 广东省农业科学院作物研究所 | Plant total protein extracting solution and application thereof |
| CN101985461A (en) * | 2010-09-19 | 2011-03-16 | 生工生物工程(上海)有限公司 | Method for extracting total proteins from formaldehyde-fixed tissues or formaldehyde-fixed paraffin-embedded tissues |
| CN105602885A (en) * | 2015-11-18 | 2016-05-25 | 扬州大学 | Method for separating and purifying plant tapetum tissue |
| WO2016166047A1 (en) * | 2015-04-14 | 2016-10-20 | Phyture Biotech, S.L. | Cell-free plant cell culture suspension supernatant with re-youth activity and/or wound healing activity over skin cells |
| CN106939032A (en) * | 2017-03-24 | 2017-07-11 | 艾比玛特医药科技（上海）有限公司 | A kind of extract solution and extracting method for rapidly and efficiently extracting plant total protein |
-
2017
- 2017-10-19 CN CN201710976855.1A patent/CN107522771A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1803828A (en) * | 2005-12-28 | 2006-07-19 | 南方医科大学 | Method for extracting protein from paraffin-embedded tissue |
| CN101475634A (en) * | 2009-02-05 | 2009-07-08 | 中国科学院植物研究所 | Extracting method for plant chloroplast total protein and special extract therefor |
| CN101696238A (en) * | 2009-10-27 | 2010-04-21 | 广东省农业科学院作物研究所 | Plant total protein extracting solution and application thereof |
| CN101985461A (en) * | 2010-09-19 | 2011-03-16 | 生工生物工程(上海)有限公司 | Method for extracting total proteins from formaldehyde-fixed tissues or formaldehyde-fixed paraffin-embedded tissues |
| WO2016166047A1 (en) * | 2015-04-14 | 2016-10-20 | Phyture Biotech, S.L. | Cell-free plant cell culture suspension supernatant with re-youth activity and/or wound healing activity over skin cells |
| CN105602885A (en) * | 2015-11-18 | 2016-05-25 | 扬州大学 | Method for separating and purifying plant tapetum tissue |
| CN106939032A (en) * | 2017-03-24 | 2017-07-11 | 艾比玛特医药科技（上海）有限公司 | A kind of extract solution and extracting method for rapidly and efficiently extracting plant total protein |
Non-Patent Citations (3)
| Title |
|---|
| ZHIJIAN JIANG 等: "Development of an efficient protein extraction method compatible with LC-MS/MS for proteome mapping in two Australia Seagrasses Zostera muelleri and Posidonia australis", 《FRONT. PLANT SCI.》 * |
| 方福德 等: "《现代医学实验技巧全书 上》", 31 December 1995, 北京医科大学 中国协和医科大学联合出版社 * |
| 胡适宜: "实验三 花药绒毡层膜的分离及绒毡层膜用于光镜和电镜观察的样品制作方法", 《植物学通报》 * |
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Application publication date: 20171229 |