CN100541185C - The high-resolution separation method of jatropha curcas seed endosperm protein - Google Patents
The high-resolution separation method of jatropha curcas seed endosperm protein Download PDFInfo
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- CN100541185C CN100541185C CNB2007100870315A CN200710087031A CN100541185C CN 100541185 C CN100541185 C CN 100541185C CN B2007100870315 A CNB2007100870315 A CN B2007100870315A CN 200710087031 A CN200710087031 A CN 200710087031A CN 100541185 C CN100541185 C CN 100541185C
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Abstract
The invention discloses the high-resolution separation method of a kind of jatropha curcas seed endosperm protein, comprise preparation contain the sample buffer of albumen dry powder, first to isoelectric focusing polyacrylamide gel electrophoresis, running gel bar balance, second to steps such as sds polyacrylamide gel electrophoresis, running gel dyeing and the graphical analyses of gel scanner uni.The present invention adopts dielectrophoresis to overcome conventional H PLC partition method etc. can not be with the protein mixed liquor by isoelectric point, molecular weight separately and can't know shortcoming such as each protein abundance, create the high-resolution isolation technics of jatropha curcas seed endosperm protein, can present isoelectric point and the molecular weight and the expression of each protein clear, intuitively.
Description
One, technical field
The present invention relates to a kind of vegetable seed protein white matter separation method, more particularly relate to the high-resolution separation method of a kind of jatropha curcas seed endosperm protein.
Two, background technology
Jatropha curcas (Jatropha curcas L.) is the inferior magaphanerophytes of the perennial fallen leaves of Euphorbiaceae leprosy Pterostyrax, and growth is fast, can yield positive results then, and seed contains innage, also has drought-resistant, impoverishment tolerant, strong stress resistance.Along with increasingly sharpening of energy crisis and problem of environmental pollution, the development and use of regenerative resource are extremely urgent.These advantages of Jatropha curcas make it become seeds that quilt is quite had an optimistic view of in the biomass energy research just.The United Nations is used for poor dry-hot valley area ecological construction with Jatropha curcas and extract thereof, and is helped as the major project of poverty alleviation.Economize the severe water and soil erosion zone in China Sichuan, Yunnan etc., Jatropha curcas is at the solid soil of water conservation, anti-ly desertifies, increases organic soil property, builds aspect such as shelter-forest and brought into play vital role, and classified as the important species of development renewable sources of energy biodiesel by country.Jatropha curcas seed contains a large amount of oil contents, except that mainly doing the bio-fuel, also can do soap, cosmetics, contains the various active composition simultaneously and can be used for agricultural chemicals and field of medicaments.Therefore the Jatropha curcas that is distributed widely in the tropical and subtropical zone area is that collection environmental protection, energy construction, ecological construction, medical product, poverty alleviation etc. are the perennial woody of one.
Jatropha curcas seed contains a lot of useful proteins matter, more to its research is a kind of curcin matter (Curcin), claim ribosome inactivating protein (RIPs) again, it has the effect that suppresses eukaryotic cell free system protein synthesis, tumor and cancer, fungal disease there are stronger inhibition and kill capability, in clinical medicine and agricultural application, show tempting prospect.
The Jatropha curcas endosperm accounts for more than 55% of seed weight, is the significant points of useful components such as storage fat, albumen, and its protein content accounts for kind of more than 25% of benevolence dry weight.But its protein component and kind are known little about it so far, and for excellent research jatropha curcas seed biological question, its economic worth of development and utilization must be set up the high-resolution separation method of jatropha curcas seed endosperm protein efficiently.
Three, summary of the invention
The purpose of this invention is to provide the high-resolution separation method of a kind of jatropha curcas seed endosperm protein.
These and other purpose of the present invention will further embody and set forth by following detailed description and explanation.
The high-resolution separation method of jatropha curcas seed endosperm protein of the present invention comprises that preparation contains the sample buffer of albumen dry powder, first and dyes and gel scanner uni image analysis step to sds polyacrylamide gel electrophoresis, running gel to isoelectric focusing polyacrylamide gel electrophoresis, running gel bar balance, second.
In the high-resolution separation method of jatropha curcas seed endosperm protein of the present invention, the sample buffer that described preparation contains albumen dry powder is 0.5-1.5 milligram albumen dry powder to be dissolved in 50-150 μ L contain in the sample buffer of 9M urea, 4% Nonidet P40,2%Ampholine ampholyte and 5% mercaptoethanol, surplus deionized water.
In the high-resolution separation method of jatropha curcas seed endosperm protein of the present invention, described first is that preparation contains 3.6% polyacrylamide gel to isoelectric focusing polyacrylamide gel electrophoresis step, 8M urea, 2% Nonidet P40, each 2.5%Ampholine ampholyte, the surplus deionized water, the gel of pH3.5-10 and pH5-8, being injected into internal diameter is that 3mm is highly in the glass tube of 15cm, the gel height is 13cm, on inject the high distilled water of about 0.5cm, quiet putting treated more than 2 hours after the complete polymerization of gel, blot the distilled water at gel column top, glass tube is installed on the electrophoresis mortise.Get jatropha curcas seed endosperm protein 30 μ l and be added on gel upper end, again with 1/2 sample buffer covering sample.Sample end (upper end) adds 0.02N NaOH ability cathode electrophoresis damping fluid, the other end (lower end) adds 0.02N phosphoric acid positive pole electrophoretic buffer, being communicated with power supply is electrophoresis after 30 minutes under the 200V condition at voltage, be raised to 400V voltage electrophoresis 15 hours, rise the isoelectric focusing that the 800V electrophoresis carried out protein in 1 hour at last.
In the high-resolution separation method of jatropha curcas seed endosperm protein of the present invention, described running gel bar equilibrium step is after isoelectric focusing is finished, gel is taken out in glass tube, place the equilibrium liquid that contains 50mM Tris-HCl, 10% glycerine, 2.5%SDS, 5% mercaptoethanol, on 60 times/minute shaking table, shook 15 minutes, and repeat 1 time.
In the high-resolution separation method of jatropha curcas seed endosperm protein of the present invention, described second is that earlier the preparation polyacrylamide is respectively the vertical panel glue that 15% separation gel and 4% concentrates glue to the sds polyacrylamide gel electrophoresis step, the upper end that then that balance is good adhesive tape is transferred to, agarose sealing with 1%, place on the electrophoresis tank, filling it up with the electrode buffer that contains 0.3%Tris, 1.44% glycocoll, 0.1%SDS, surplus deionized water, is about 3 hours of electrophoresis under the condition of 30mA at electric current.
In the high-resolution separation method of jatropha curcas seed endosperm protein of the present invention, described running gel staining procedure be second after the sds polyacrylamide gel electrophoresis step is finished, take off gel, place colouration box to add the immobile liquid that 100ml contains 40% ethanol, 10% glacial acetic acid and surplus deionized water, on 60 times/minute shaking table, shook 1 hour, change the dyeing liquor that 100ml contains 0.116% Coomassie brilliant blue R-250,25% ethanol, 8% glacial acetic acid and surplus deionized water, on 60 times/minute shaking table, shook 1 hour; Pour out dyeing liquor, add the destainer that 100ml contains 25% ethanol, 8% glacial acetic acid and surplus deionized water, on 60 times/minute shaking table, shook 30 minutes, repeat 3 times, obtain the distinct gel electrophoresis spectrum (as shown in Figure 1) of protein site.
In the high-resolution separation method of jatropha curcas seed endosperm protein of the present invention, described gel scanner uni image analysis step is to be 256 rank gray scales, 600dpi condition under transmission scan with the UMAX scanner in parameter with the gel behind the coomassie brilliant blue staining, gained image ImageMaster
TM2D Platinum software (Amersham Bioscience) is analyzed, the result shows, can detect protein site on the gel and have 1 at least, more than 000, it is the 35-65kDa zone for 4.5-6, molecular weight that the distribution of its protein mainly concentrates on isoelectric point (pI), be the protein monoid of some subaciditys, intermediate molecular weight, more than 20 high abundance expressed protein (as shown in Figure 2) arranged at the low-molecular-weight area discover.
The present invention adopts dielectrophoresis to overcome conventional H PLC partition method etc. can not be with the protein mixed liquor by isoelectric point, molecular weight separately and can't know shortcoming such as each protein abundance, created the high-resolution isolation technics of jatropha curcas seed endosperm protein, isoelectric point and the molecular weight and the expression that can present each protein clear, intuitively, being that other separation method is incomparable, is most important, one of the most effective means of proteomics research.And because first be from tubulation glue to isoelectric focusing, the pH gradient can be modulated voluntarily, and the while low price has great application prospect in scientific research and production practices.
In this application, can use the commercially available jatropha curcas seed endosperm protein that is used for electrophoresis, also can use the exercise question of applying on the same day with the application to be: the jatropha curcas seed endosperm protein that a kind of method for extracting jatropha curcas seed endosperm protein extracted, all the elements of this patented claim are listed use, reference at this.
Four, description of drawings
Fig. 1 is a jatropha curcas seed protein dielectrophoresis proteomic expression profile.
Fig. 2 is a jatropha curcas seed proteomic expression profile digital imagery analysis result.
Five, embodiment
Below further specify the present invention by specific embodiment, but embodiment only be used for the explanation, can not limit the scope of the invention.
In the present invention, if not refer in particular to, all parts, amount are the unit of weight based on general assembly (TW), and all raw materials all can be buied from market, form in the liquid at each, and surplus is a deionized water.
Embodiment 1
Carry out by the following method:
1, preparation contains the sample buffer of albumen dry powder:
With 1 milligram of albumen dry powder be dissolved in contain 100 microlitre 9M urea, 4% Nonidet P40,2%Ampholine ampholyte, in the sample buffer of 5% mercaptoethanol and surplus deionized water.
2, first to the isoelectric focusing polyacrylamide gel electrophoresis:
Preparation contains the gel of 3.6% polyacrylamide gel, 8M urea, 2% Nonidet P40, each 2.5%Ampholine ampholyte pH3.5-10 and pH5-8, being injected into internal diameter is that 3mm is highly in the glass tube of 15cm, the gel height is 13cm, on inject the high distilled water of about 0.5cm, quiet putting treated more than 2 hours after the complete polymerization of gel, blot the distilled water at gel column top, glass tube is installed on the electrophoresis mortise.Get jatropha curcas seed endosperm protein 30 μ l and be added on gel upper end, again with 1/2 sample buffer covering sample.Sample end (upper end) adds 0.02N NaOH ability cathode electrophoresis damping fluid, the other end (lower end) adds 0.02N phosphoric acid positive pole electrophoretic buffer, being communicated with power supply is electrophoresis after 30 minutes under the 200V condition at voltage, be raised to 400V voltage electrophoresis 15 hours, rise the isoelectric focusing that the 800V electrophoresis carried out protein in 1 hour at last.
3, running gel bar balance:
After isoelectric focusing is finished, gel is taken out in glass tube, place the equilibrium liquid that contains 50mMTris-HCl, 10% glycerine, 2.5%SDS, 5% mercaptoethanol, on 60 times/minute shaking table, shook 15 minutes, and repeat 1 time.
4, second to sds polyacrylamide gel electrophoresis:
The preparation polyacrylamide is respectively the vertical panel glue that 15% separation gel and 4% concentrates glue earlier, the upper end that then that balance is good adhesive tape is transferred to, agarose sealing with 1%, place on the electrophoresis tank, filling it up with the electrode buffer that contains 0.3%Tris, 1.44% glycocoll, 0.1%SDS and surplus deionized water, is about 3 hours of electrophoresis under the condition of 30mA at electric current.
5, running gel staining procedure:
Second after the sds polyacrylamide gel electrophoresis step is finished, take off gel, place colouration box to add the immobile liquid that 100ml contains 40% ethanol, 10% glacial acetic acid and surplus deionized water, on 60 times/minute shaking table, shook 1 hour, change the dyeing liquor that 100ml contains 0.116% Coomassie brilliant blue R-250,25% ethanol, 8% glacial acetic acid and surplus deionized water, on 60 times/minute shaking table, shook 1 hour; Pour out dyeing liquor, add the destainer that 100ml contains 25% ethanol, 8% glacial acetic acid and surplus deionized water, on 60 times/minute shaking table, shook 30 minutes, repeat 3 times, obtain the distinct gel electrophoresis spectrum (as shown in Figure 1) of protein site.
6, gel scanner uni graphical analysis:
Is 256 rank gray scales, 600dpi condition under transmission scan with the UMAX scanner in parameter with the gel behind the coomassie brilliant blue staining, gained image ImageMaster
TM2D Platinum software (Amersham Bioscience) is analyzed, the result shows, can detect protein site on the gel and have 1 at least, more than 000, it is the 35-65kDa zone for 4.5-6, molecular weight that the distribution of its protein mainly concentrates on isoelectric point (pI), be the protein monoid of some subaciditys, intermediate molecular weight, more than 20 high abundance expressed protein (as shown in Figure 2) arranged at the low-molecular-weight area discover.
Embodiment 2
Extract as follows:
A, get 200 milligrams of air-dry jatropha curcas seed endosperms of maturation, grind to form meticulous powder (particle diameter is about 0.1mm) with mortar, add 2 milliliters of precooling homogenate buffers then, this damping fluid contains the poly-ethoxy ethanol X-100 of 20mM Tris-HCl, 250mM sucrose, 10mM ethylenediamine tetraacetic acid, 1.0mM two sulphur uncle sugar alcohols and 1% Octylphenoxy, surplus is a deionized water.Be ground to homogenate (milling time is 10 minutes, and rotating speed is about 120 rev/mins) on ice;
B, homogenate is transferred in the centrifuge tube, under 4 ℃ centrifugal 15 minutes in 15000g, abandon precipitation, in supernatant, add the trichloroacetic acid of 10% (v/v), then precipitation 40 minutes in ice bath;
C, with B step the same terms under centrifugal once more after, remove supernatant, it is centrifugal that precipitation is cleaned the back with acetone, gets precipitation again;
D, repeat to clean 2-3 time with acetone, under 4 ℃ centrifugal 40 minutes in 15000g, remove supernatant, will precipitate vacuum drying and obtain albumen dry powder.
Claims (1)
1, the high-resolution separation method of a kind of jatropha curcas seed endosperm protein, it is characterized in that comprising sample buffer step, first that preparation contains albumen dry powder to isoelectric focusing polyacrylamide gel electrophoresis step, running gel bar equilibrium step, second to sds polyacrylamide gel electrophoresis step, running gel staining procedure and gel scanner uni image analysis step;
The sample buffer that wherein said preparation contains albumen dry powder is 0.5-1.5 milligram albumen dry powder to be dissolved in 50-150 μ l contain in the sample buffer of 9M urea, 4% Nonidet P40,2%Ampholine ampholyte and 5% mercaptoethanol;
Described first is that preparation contains 3.6% polyacrylamide gel to isoelectric focusing polyacrylamide gel electrophoresis step, 8M urea, 2% Nonidet P40, each 2.5%Ampholine ampholyte, the gel of pH3.5-10 and pH5-8, being injected into internal diameter is that 3mm is highly in the glass tube of 15cm, the gel height is 13cm, on inject the high distilled water of about 0.5cm, quiet putting treated more than 2 hours after the complete polymerization of gel, blot the distilled water at gel column top, glass tube is installed on the electrophoresis mortise, get jatropha curcas seed endosperm protein 30 μ l and be added on gel upper end, again with 1/2 sample buffer covering sample; The sample end adds 0.02N NaOH ability cathode electrophoresis damping fluid, the other end adds 0.02N phosphoric acid positive pole electrophoretic buffer, being communicated with power supply is that electrophoresis was raised to 400V voltage electrophoresis 15 hours after 30 minutes under the 200V condition at voltage, rises the isoelectric focusing that the 800V electrophoresis carried out protein in 1 hour at last;
Described running gel bar equilibrium step is after isoelectric focusing is finished, gel is taken out in glass tube, place the equilibrium liquid that contains 50mM Tris-HCl, 10% glycerine, 2.5%SDS, 5% mercaptoethanol, on 60 times/minute shaking table, shook 15 minutes, and repeat 1 time;
Described second is that earlier the preparation polyacrylamide is respectively the vertical panel glue that 15% separation gel and 4% concentrates glue to the sds polyacrylamide gel electrophoresis step, then that balance is good adhesive tape is transferred to the upper end of the vertical hectograph of electrophoresis tank, agarose sealing with 1%, place on the electrophoresis tank, filling it up with the electrode buffer that contains 0.3%Tris, 1.44% glycocoll, 0.1%SDS, is about 3 hours of electrophoresis under the condition of 30mA at electric current;
Described running gel staining procedure be second after the sds polyacrylamide gel electrophoresis step is finished, take off gel, place colouration box to add the immobile liquid that 100ml contains 40% ethanol, 10% glacial acetic acid, on 60 times/minute shaking table, shook 1 hour, change the dyeing liquor that 100ml contains 0.116% Coomassie brilliant blue R-250,25% ethanol, 8% glacial acetic acid, on 60 times/minute shaking table, shook 1 hour; Pour out dyeing liquor, add the destainer that 100ml contains 25% ethanol, 8% glacial acetic acid, on 60 times/minute shaking table, shook 30 minutes, repeat 3 times, obtain the distinct gel electrophoresis spectrum of protein site;
Described gel scanner uni image analysis step is to be 256 rank gray scales, 600dpi condition under transmission scan with the UMAX scanner in parameter with the gel behind the coomassie brilliant blue staining, gained image ImageMaster
TM2D Platinum software is analyzed.
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| CN101718745B (en) * | 2009-12-07 | 2012-10-10 | 南京农业大学 | Bidirectional electrophoresis method for total protein of jute root system |
| CN104004382B (en) * | 2014-05-20 | 2016-01-20 | 北京五康新兴科技有限公司 | A kind of coomassie brilliant blue staining liquid and dyeing process |
| CN110320086B (en) * | 2019-07-09 | 2020-04-14 | 上海宝藤生物医药科技股份有限公司 | Lipoprotein electrophoresis staining solution and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85107875A (en) * | 1985-05-21 | 1987-04-08 | 武田药品工业株式会社 | Protein is separated from each other |
| EP0568001A2 (en) * | 1992-04-27 | 1993-11-03 | Showa Shell Sekiyu Kabushiki Kaisha | Antiviral agent containing crude drug |
| AT406674B (en) * | 1997-12-17 | 2000-07-25 | Sucher & Holzer Bauplan Handel | Organic complex compound and anti-inflammatory medicament |
| CN1226418C (en) * | 2002-07-12 | 2005-11-09 | 广东欧瑞卡生物工程有限公司 | Natural active protein and peptide separating and purifying process |
| CN1714863A (en) * | 2005-05-31 | 2006-01-04 | 四川大学 | Method for extracting curcin and its use |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85107875A (en) * | 1985-05-21 | 1987-04-08 | 武田药品工业株式会社 | Protein is separated from each other |
| EP0568001A2 (en) * | 1992-04-27 | 1993-11-03 | Showa Shell Sekiyu Kabushiki Kaisha | Antiviral agent containing crude drug |
| AT406674B (en) * | 1997-12-17 | 2000-07-25 | Sucher & Holzer Bauplan Handel | Organic complex compound and anti-inflammatory medicament |
| CN1226418C (en) * | 2002-07-12 | 2005-11-09 | 广东欧瑞卡生物工程有限公司 | Natural active protein and peptide separating and purifying process |
| CN1714863A (en) * | 2005-05-31 | 2006-01-04 | 四川大学 | Method for extracting curcin and its use |
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