CN105481941A - Plant protein extraction method and plant proteomic analysis method - Google Patents
Plant protein extraction method and plant proteomic analysis method Download PDFInfo
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- CN105481941A CN105481941A CN201610041031.0A CN201610041031A CN105481941A CN 105481941 A CN105481941 A CN 105481941A CN 201610041031 A CN201610041031 A CN 201610041031A CN 105481941 A CN105481941 A CN 105481941A
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2866—Grinding or homogeneising
Abstract
The invention discloses a plant protein extraction method and a plant proteomic analysis method. The plant protein extraction method comprises steps as follows: (1) plant samples are ground into powder and mixed with an extraction solution, grinding balls are put in the mixed solution, and the mixture is vibrated and left to stand; (2), the mixed solution left to stand is centrifuged, a supernatant is discarded, precipitates are washed with a first acetone solution to be pure white, a second acetone solution is used for purification, the purified precipitates are subjected to vacuum freeze drying, and protein dry powder is obtained. According to the method, the problems of insufficient extraction of protein of plant seeds and high extraction impurity content in the prior art are solved, the plant seed protein is more sufficiently extracted, the extraction purity is higher, and the protein purification cost and time are saved.
Description
Technical field
The present invention relates to biological technical field, be specifically related to extracting method and the plant proteomics research analytical procedure of a kind of plant protein.
Background technology
Proteomics research is in proteomics level, systematic research is goed deep into the function perform bulk-protein of vital movement, not only contribute to the essence disclosing vital movement panoramically, and all significant for study of disease mechanism, development early warning, Diagnosis and Treat method.And the proteins extraction of animals and plants sample is the steps necessary of proteomics research.In proteomics research, sample preparation is the step of most critical, directly determines the success or failure of subsequent experimental.When extracting the protein of the especially high lipid seed of plant seed, employing ordinary method not only can not be abundant by proteins extraction, and the protein impurities content extracted is high, can not obtain highly purified protein; And the insufficient meeting of proteins extraction causes the low abundance proteins performing important biomolecule function to be lost, the failure that protein impurities content height then often causes protein first to be tested to isoelectrofocusing, second can not obtain protein spots clearly to vertical slab electrophoresis, finally causes the failure of whole experiment.In order to obtain highly purified protein, also can adopt protein purification test kit, but price is general costly.
Summary of the invention
The embodiment of the present application is by providing extracting method and the plant proteomics research analytical procedure of a kind of plant protein, solve vegetable seed protein white matter in prior art extract insufficient and extract the high problem of foreign matter content, to the extraction of vegetable seed protein white matter is more abundant and DNA purity is higher, and purify protein expense and time can not only be saved.
For achieving the above object, the present invention mainly provides following technical scheme:
On the one hand, embodiments provide the extracting method of a kind of plant protein, comprise the following steps:
(1) by plant sample grind into powder, then mix with extracting solution, vibrate put into grinding bead in mixed solution after, then leave standstill;
(2) by centrifugal for the mixed solution after leaving standstill, abandon supernatant liquor, precipitation first adopted the first acetone soln washing to pure white, then adopt the second acetone soln to purify, by the precipitation vacuum lyophilization after purification, namely obtain protein dry powder.
As preferably, described plant sample is plant seed.
As preferably, described plant sample is high lipid plant seed.
As preferably, when carrying out described washing in described step (2), first in precipitation, add the first acetone soln, then vibrate, leave standstill, centrifugal, to the precipitation after centrifugal according to identical method repeated washing, until be precipitated as pure white.
As preferably, when carrying out described purification in described step (2), first in pure white precipitation, add the second acetone soln, then vibrate, leave standstill, centrifugal, the centrifugal precipitation obtained is the precipitation after purification.
As preferably, when carrying out described vibration, mixed solution and grinding bead are put into grinding pot or centrifuge tube, then grinding pot or centrifuge tube are put into beveller, adopt beveller to vibrate.
As preferably, the diameter of described grinding bead is the 1/3-2/3 of grinding pot diameter or centrifuge tube nozzle diameter, and the quantity of grinding bead is 1-2.
As preferably, the frequency of described vibration is 1500-2500 beat/min.
As preferably, the time of described vibration is 2-10min.
As preferably, the material of described grinding bead is selected from the one in zirconium dioxide, converted steel, stainless steel, wolfram varbide, agate or tynex; The material of described grinding pot is selected from the one in zirconium dioxide, converted steel, stainless steel, wolfram varbide, agate or tynex; The material of described centrifuge tube is selected from the one in zirconium dioxide, converted steel, stainless steel, wolfram varbide, agate or tynex.
On the other hand, the embodiment of the present invention additionally provides a kind of plant proteomics research analytical procedure, comprises the extraction to plant protein, and the described extraction to plant protein adopts above-mentioned extracting method.
The one or more technical schemes provided in the embodiment of the present application, at least have following technique effect or advantage:
The embodiment of the present invention is vibrated by adopting beveller to extract mixed solution to plant sample, plant sample fully can be contacted with extracting solution, protein in plant sample is fully precipitated and fully discharges the impurity be wrapped in particle, thus make the extraction of protein more fully and the foreign matter content decreased in the protein of extraction, avoid and use expensive protein purification test kit, save purify protein expense; The time vibrated proteins extraction mixed solution is short and easy and simple to handle, cost is lower, saves extraction time and extraction cost.
Accompanying drawing explanation
Fig. 1 is the Two-dimensional Gel Electrophoresis of the protein that the embodiment of the present invention and comparative example are extracted.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, but not as a limitation of the invention.
Embodiment 1
(1) take 0.25g to shell cotton seeds, add 0.05g polyvinylpyrrolidone (PVP) and liquid nitrogen, rapid grind into powder, load in 2mL centrifuge tube, TCA (the trichoroacetic acid(TCA))-acetone extract adding 1.2mL-20 DEG C of precooling again mixes, this extracting solution is be the TCA of 10% containing massfraction, massfraction be 0.07% beta-mercaptoethanol and volumetric molar concentration be the acetone soln of the phenylmethylsulfonyl fluoride of 1mmol/L, wherein the volume of extracting solution is 3 times of cotton seeds powder volume, put into 1 grinding bead in mixed solution after, centrifuge tube is placed in beveller and vibrates 2min, be placed in the environment hold over night of-20 DEG C again, vibrate therebetween repeatedly, wherein the material of centrifuge tube is polypropylene, the material of grinding bead is zirconium dioxide, grinding bead diameter is 5mm, vibrational frequency is 1800 beats/min,
(2) by the mixed solution after above-mentioned hold over night under the condition of 4 DEG C with the centrifugal force 30min of 25000 × g, abandon supernatant liquor, the first acetone soln mixing of 1.2mL-20 DEG C of precooling is added in the precipitation in centrifuge tube, this first acetone soln be containing massfraction be 0.07% beta-mercaptoethanol and volumetric molar concentration be the acetone soln of the phenylmethylsulfonyl fluoride of 1mmol/L, centrifuge tube is placed in beveller and vibrates 2min (now grinding bead is still in centrifuge tube), vibrational frequency is 1800 beats/min, the environment being placed in-20 DEG C again leaves standstill 1h, then under the condition of 4 DEG C with the centrifugal force 30min of 25000 × g, to the precipitation after centrifugal according to identical method repeated washing, until be precipitated as pure white, the second acetone soln of 1.2mL-20 DEG C of precooling is added again in the pure white precipitation in centrifuge tube, this second acetone soln to be acetone volume fraction be 80% aqueous acetone solution, then centrifuge tube is placed in beveller and vibrates 2min (now grinding bead is still in centrifuge tube), vibrational frequency is 1800 beats/min, the environment being placed in-20 DEG C again leaves standstill 1h, with the centrifugal force 30min of 25000 × g under the condition of 4 DEG C, finally the centrifugal precipitation vacuum lyophilization obtained is become dry powder, this dry powder is cotton seeds protein dry powder, save backup or carry out subsequent operations.
Embodiment 2
(1) 0.25g oil sunflower is taken, add 0.05g polyvinylpyrrolidone (PVP) and liquid nitrogen, rapid grind into powder, load in 2mL centrifuge tube, the TCA-acetone extract adding 1.2mL-20 DEG C of precooling again mixes, this extracting solution is be the TCA of 10% containing massfraction, massfraction be 0.07% beta-mercaptoethanol and volumetric molar concentration be the acetone soln of the phenylmethylsulfonyl fluoride of 1mmol/L, wherein the volume of extracting solution is 3 times of oil sunflower powder volume, put into 1 grinding bead in mixed solution after, centrifuge tube is placed in beveller and vibrates 5min, be placed in the environment hold over night of-20 DEG C again, vibrate therebetween repeatedly, wherein the material of centrifuge tube is polypropylene, the material of grinding bead is wolfram varbide, grinding bead diameter is 5mm, vibrational frequency is 2500 beats/min,
(2) by the mixed solution after above-mentioned hold over night under the condition of 4 DEG C with the centrifugal force 30min of 25000 × g, abandon supernatant liquor, the first acetone soln mixing of 1.2mL-20 DEG C of precooling is added in the precipitation in centrifuge tube, this first acetone soln be containing massfraction be 0.07% beta-mercaptoethanol and volumetric molar concentration be the acetone soln of the phenylmethylsulfonyl fluoride of 1mmol/L, centrifuge tube is placed in beveller and vibrates 5min (now grinding bead is still in centrifuge tube), vibrational frequency is 2500 beats/min, the environment being placed in-20 DEG C again leaves standstill 1h, then under the condition of 4 DEG C with the centrifugal force 30min of 25000 × g, to the precipitation after centrifugal according to identical method repeated washing, until be precipitated as pure white, the second acetone soln of 1.2mL-20 DEG C of precooling is added again in the pure white precipitation in centrifuge tube, this second acetone soln to be acetone volume fraction be 80% aqueous acetone solution, then centrifuge tube is placed in beveller and vibrates 5min (now grinding bead is still in centrifuge tube), vibrational frequency is 2500 beats/min, the environment being placed in-20 DEG C again leaves standstill 1h, with the centrifugal force 30min of 25000 × g under the condition of 4 DEG C, finally the centrifugal precipitation vacuum lyophilization obtained is become dry powder, this dry powder is oil sunflower protein dry powder, save backup or carry out subsequent operations.
Embodiment 3
(1) 6.25g peanut seed is taken, add 1.25g polyvinylpyrrolidone (PVP) and liquid nitrogen, rapid grind into powder, load in 50mL grinding pot, the TCA-acetone extract adding 30mL-20 DEG C of precooling again mixes, this extracting solution is be the TCA of 10% containing massfraction, massfraction be 0.07% beta-mercaptoethanol and volumetric molar concentration be the acetone soln of the phenylmethylsulfonyl fluoride of 1mmol/L, wherein the volume of extracting solution is 3 times of peanut seed powder volume, put into 2 grinding bead in mixed solution after, grinding pot is placed in beveller and vibrates 10min, be placed in the environment hold over night of-20 DEG C again, vibrate therebetween repeatedly, wherein the material of grinding pot is stainless steel, the material of grinding bead is stainless steel, grinding bead diameter is 10mm, vibrational frequency is 1500 beats/min,
(2) mixed solution after above-mentioned hold over night is transferred in centrifuge tube together with grinding bead, and with the centrifugal force 30min of 25000 × g under the condition of 4 DEG C, abandon supernatant liquor, the first acetone soln mixing of 30mL-20 DEG C of precooling is added in the precipitation in centrifuge tube, this first acetone soln be containing massfraction be 0.07% beta-mercaptoethanol and volumetric molar concentration be the acetone soln of the phenylmethylsulfonyl fluoride of 1mmol/L, mixed solution in centrifuge tube and grinding bead are transferred in grinding pot, and be placed in beveller and vibrate 10min, vibrational frequency is 1500 beats/min, the environment being placed in-20 DEG C again leaves standstill 1h, then mixed solution is transferred in centrifuge tube together with grinding bead, and with the centrifugal force 30min of 25000 × g under the condition of 4 DEG C, to the precipitation after centrifugal according to identical method repeated washing, until be precipitated as pure white, the second acetone soln of 30mL-20 DEG C of precooling is added again in the pure white precipitation in centrifuge tube, this second acetone soln to be acetone volume fraction be 80% aqueous acetone solution, then the mixed solution in centrifuge tube and grinding bead are transferred in grinding pot, and be placed in beveller and vibrate 10min, vibrational frequency is 1500 beats/min, the environment being placed in-20 DEG C again leaves standstill 1h, then mixed solution is transferred in centrifuge tube together with grinding bead, and with the centrifugal force 30min of 25000 × g under the condition of 4 DEG C, finally the centrifugal precipitation vacuum lyophilization obtained is become dry powder, this dry powder is peanut seed protein dry powder, save backup or carry out subsequent operations.
Embodiment 4
(1) 6.25g soybean seeds is taken, add 1.25g polyvinylpyrrolidone (PVP) and liquid nitrogen, rapid grind into powder, load in 50mL grinding pot, the TCA-acetone extract adding 30mL-20 DEG C of precooling again mixes, this extracting solution is be the TCA of 10% containing massfraction, massfraction be 0.07% beta-mercaptoethanol and volumetric molar concentration be the acetone soln of the phenylmethylsulfonyl fluoride of 1mmol/L, wherein the volume of extracting solution is 3 times of soybean seeds powder volume, put into 2 grinding bead in mixed solution after, grinding pot is placed in beveller and vibrates 8min, be placed in the environment hold over night of-20 DEG C again, vibrate therebetween repeatedly, wherein the material of grinding pot is agate, the material of grinding bead is agate, grinding bead diameter is 10mm, vibrational frequency is 2000 beats/min,
(2) mixed solution after above-mentioned hold over night is transferred in centrifuge tube together with grinding bead, and with the centrifugal force 30min of 25000 × g under the condition of 4 DEG C, abandon supernatant liquor, the first acetone soln mixing of 30mL-20 DEG C of precooling is added in the precipitation in centrifuge tube, this first acetone soln be containing massfraction be 0.07% beta-mercaptoethanol and volumetric molar concentration be the acetone soln of the phenylmethylsulfonyl fluoride of 1mmol/L, mixed solution in centrifuge tube and grinding bead are transferred in grinding pot, and be placed in beveller and vibrate 8min, vibrational frequency is 2000 beats/min, the environment being placed in-20 DEG C again leaves standstill 1h, then mixed solution is transferred in centrifuge tube together with grinding bead, and with the centrifugal force 30min of 25000 × g under the condition of 4 DEG C, to the precipitation after centrifugal according to identical method repeated washing, until be precipitated as pure white, the second acetone soln of 30mL-20 DEG C of precooling is added again in the pure white precipitation in centrifuge tube, this second acetone soln to be acetone volume fraction be 80% aqueous acetone solution, then the mixed solution in centrifuge tube and grinding bead are transferred in grinding pot, and be placed in beveller and vibrate 8min, vibrational frequency is 2000 beats/min, the environment being placed in-20 DEG C again leaves standstill 1h, then mixed solution is transferred in centrifuge tube together with grinding bead, and with the centrifugal force 30min of 25000 × g under the condition of 4 DEG C, finally the centrifugal precipitation vacuum lyophilization obtained is become dry powder, this dry powder is soybean seed protein matter dry powder, save backup or carry out subsequent operations.
Comparative example 1
This comparative example cotton seeds that shelled by 0.25g conventionally extracts protein, this ordinary method is compared with the method for embodiment 1, except not putting into grinding bead in centrifuge tube, with centrifuge tube is placed in beveller and vibrates 2min and change into and centrifuge tube is placed in vortex instrument and vibrates 2min, vortex instrument oscillation frequency is outside 1800rpm/min, and all the other operation stepss are all with embodiment 1.
Comparative example 2
0.25g oil sunflower is conventionally extracted protein by this comparative example, this ordinary method is compared with the method for embodiment 2, except not putting into grinding bead in centrifuge tube, with centrifuge tube is placed in beveller and vibrates 5min and change into centrifuge tube is placed in ultrasonic apparatus ice-bath ultrasonic, and ultrasonic apparatus power is 400w, ultrasonic 5s again interval 5s is total to outside ultrasonic 25min, and all the other operation stepss are all with embodiment 2.
Comparative example 3
6.25g peanut seed is conventionally extracted protein by this comparative example, this ordinary method is compared with the method for embodiment 3, beveller is adopted to vibrate except mixed solution and grinding bead are put into grinding pot, change into and do not put into grinding bead in mixed solution, directly be placed in centrifuge tube by mixture outside fully stirring it, all the other operation stepss are all with embodiment 3.
Comparative example 4
6.25g soybean seeds is conventionally extracted protein by this comparative example, this ordinary method is compared with the method for embodiment 4, except not putting into grinding bead in grinding pot, with grinding pot is placed in beveller and vibrates 8min and change into and grinding pot is placed in vortex instrument and vibrates 8min, vortex instrument oscillation frequency is outside 1800rpm/min, and all the other operation stepss are all with embodiment 4.
Protein dry powder above-described embodiment and comparative example prepared carries out two-dimensional electrophoresis experiment in accordance with the following methods: by 1mg protein dry powder and 25 μ L lysates (the IPG damping fluid (pH is 4-7) that the urea containing 9mol/L, massfraction be the CHAPS (3-[3-(courage amido propyl) dimethylamino] propanesulfonic acid inner salt) of 4%, the DTT (dithiothreitol (DTT)) of 65mmol/L and massfraction are 0.4%) fully vortex mix; By mixed solution in 30 DEG C of water-bath 30min, more abundant cooled with liquid nitrogen after vibration mixing, repeat this water-bath and process of cooling 2 times, then by mixture under the condition of 20 DEG C with the centrifugal force 30min of 25000 × g, Aspirate supernatant, measures its protein concn; After supernatant liquor being diluted to 1 μ g/1 μ L according to the protein concn recorded, getting 5 μ L and carry out polyacrylamide gel electrophoresis experiment, obtain electrophoretic band as shown in Figure 1.Wherein standard protein is made up of 7 kinds of pre-dyed protein, and molecular weight ranges is 14kDa-100kDa, and often kind of albumen (i.e. each bands of a spectrum) is 2 μ g albumen; Wherein M
1, K
1, H
1, D
1be respectively the electrophoretic band of the protein that embodiment 1, embodiment 2, embodiment 3 and embodiment 4 are extracted, M
2, K
2, H
2, D
2be respectively the electrophoretic band of the protein that comparative example 1, comparative example 2, comparative example 3 and comparative example 4 are extracted.
As seen from Figure 1, the electrophoretic band of the different vegetable seed protein white matters that the embodiment of the present invention is extracted is compared with the electrophoretic band of corresponding comparative example, protein domain resolving power is higher, protein band form is clearer and protein distribution is wider, especially more with high molecular low-abundance protein band, and band is relatively less, albumen sepn better effects if, describe compared with common protein extracting method, the extraction of embodiment of the present invention method to protein is more abundant, the protein mass extracted is more, and foreign matter content is relatively less, both good extraction effect had been obtained, turn avoid and use expensive protein purification test kit, save purify protein expense.When extracting protein to the especially high lipid plant seed of plant seed sample, seed shells after liquid nitrogen fully grinds, be easy to stick into one in extracting solution, and it is very hard, adopting in comparative example that the vortex instrument of ordinary method vibrates, ultrasonic apparatus is ultrasonic or stirring all to make seed sample fully disperse in extracting solution, the protein in seed and extracting solution can not be made fully to contact and precipitate, thus can not high purity be extracted and the wider protein that distributes; The embodiment of the present invention adopts beveller and grinding bead fully to vibrate plant sample extraction mixed solution, glutinous protein group together is fully disperseed in extracting solution, and fully contact with extracting solution and precipitate, fully release the impurity be wrapped in sample particle simultaneously, thus after pelleting centrifugation, obtain that purity is higher, distribution is wider and the more protein of content, when this protein is used for Two-Dimensional Gel Electrophoresis experiment and protein separation qualification, all can obtain extraordinary effect; The time that the embodiment of the present invention is vibrated proteins extraction mixed solution is short and easy and simple to handle, cost is lower, namely saves extraction time, turn avoid and uses expensive protein purification test kit, save experimental expenses.
The plant sample adopted in the embodiment of the present invention is plant seed, owing to containing more lipid acid in plant seed, lastly join in extracting solution pulverizing, be easy to stick into one, and it is very hard, be not easy fully to contact with extracting solution, the protein extracting method adopting the embodiment of the present invention to provide, high purity can be obtained, distribute more extensively and the more vegetable seed protein white matter of content; Embodiment of the present invention preferred plant sample is high lipid plant seed, because high lipid plant seed fatty acid content is more, when extracting protein, seed powder more easily sticks into one in extracting solution, more be difficult to obtain high purity, distribute more extensively and the more protein of content, and the protein extracting method adopting the embodiment of the present invention to provide still can obtain good extraction effect.
The embodiment of the present invention is when extracting plant protein, grinding bead is put in extraction mixed solution, mixed solution and grinding bead are put into grinding pot or centrifuge tube, again grinding pot or centrifuge tube are put into beveller, beveller is adopted to vibrate, effectively plant powder sample fully can be disperseed in extraction mixed solution, wherein grinding bead, the material of grinding pot and centrifuge tube all can be selected from zirconium dioxide, converted steel, stainless steel, wolfram varbide, one in agate or tynex, these materials were both insoluble to extracting solution, there is again enough intensity clash into the vibration of resisting grinding bead, the diameter of the preferred grinding bead of the embodiment of the present invention is the 1/3-2/3 of grinding pot diameter or centrifuge tube nozzle diameter, when the diameter of grinding bead is within the scope of this, plant powder sample can be made fully to disperse in extracting solution, when the diameter of grinding bead is less than 1/3 of grinding pot diameter or centrifuge tube nozzle diameter, grinding bead is too little, can not produce enough dynamics makes plant sample disperse in extracting solution, when the diameter of grinding bead is greater than 2/3 of grinding pot diameter or centrifuge tube nozzle diameter, grinding bead is too large, and the limited space in grinding pot or centrifuge tube, thus cause the Oscillation Amplitude of grinding bead too little, plant sample can not be made well to disperse in extracting solution, the quantity of the preferred grinding bead of the embodiment of the present invention is 1-2.
The frequency of the vibration adopted in the embodiment of the present invention is 1500-2500 beat/min, in this range of frequency, plant sample can be made fully to disperse in extracting solution, when vibrational frequency is less than 1500 beats/min, can not produce enough dynamics makes plant powder sample disperse in extracting solution, when vibrational frequency is greater than 2500 beats/min, exceed the intensity that grinding bead, grinding pot or centrifuge tube can bear, easily make it produce wearing and tearing or destroy.
The time of the vibration that the embodiment of the present invention adopts is 2-10min, in this time range, be enough to plant sample is fully disperseed in extracting solution, time of vibration is less than 2min, time is too short, the dispersion effect of plant sample in extracting solution is poor, and when time of vibration is greater than 10min, extraction time is elongated.
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (10)
1. the extracting method of a kind of plant protein, is characterized in that, comprises the following steps:
(1) by plant sample grind into powder, then mix with extracting solution, vibrate put into grinding bead in mixed solution after, then leave standstill;
(2) by centrifugal for the mixed solution after leaving standstill, abandon supernatant liquor, precipitation first adopted the first acetone soln washing to pure white, then adopt the second acetone soln to purify, by the precipitation vacuum lyophilization after purification, namely obtain protein dry powder.
2. the extracting method of plant protein according to claim 1, is characterized in that, described plant sample is plant seed.
3. the extracting method of plant protein according to claim 2, is characterized in that, described plant sample is high lipid plant seed.
4. the extracting method of plant protein according to claim 1, it is characterized in that, when carrying out described washing in described step (2), first in precipitation, add the first acetone soln, vibrate again, leave standstill, centrifugal, to the precipitation after centrifugal according to identical method repeated washing, until be precipitated as pure white.
5. the extracting method of plant protein according to claim 1, it is characterized in that, when carrying out described purification in described step (2), first in pure white precipitation, add the second acetone soln, vibrate again, leave standstill, centrifugal, the centrifugal precipitation obtained is the precipitation after purification.
6. the extracting method of the plant protein according to claim 1,4 or 5, it is characterized in that, when carrying out described vibration, mixed solution and grinding bead are put into grinding pot or centrifuge tube, again grinding pot or centrifuge tube are put into beveller, adopt beveller to vibrate.
7. the extracting method of plant protein according to claim 6, is characterized in that, the diameter of described grinding bead is the 1/3-2/3 of grinding pot diameter or centrifuge tube nozzle diameter, and the quantity of grinding bead is 1-2.
8. the extracting method of the plant protein according to claim 1,4 or 5, is characterized in that, the frequency of described vibration is 1500-2500 beat/min.
9. the extracting method of the plant protein according to claim 1,4 or 5, is characterized in that, the time of described vibration is 2-10min.
10. a plant proteomics research analytical procedure, comprises the extraction to plant protein, it is characterized in that, the described extraction to plant protein adopts the extracting method described in any one of claim 1-9.
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| CN107880095A (en) * | 2017-11-22 | 2018-04-06 | 安徽省东博米业有限公司 | A kind of corn protein rapid extracting method |
| CN109988222A (en) * | 2019-04-12 | 2019-07-09 | 龙岩学院 | A kind of quick tea root protein extracting method |
| CN110760556A (en) * | 2019-12-03 | 2020-02-07 | 安徽联河股份有限公司 | Method for high-efficiency extraction of protein in broken rice |
| 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 |
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