CN109293733A - A kind of extracting method suitable for fresh tobacco leaves holoprotein - Google Patents
A kind of extracting method suitable for fresh tobacco leaves holoprotein Download PDFInfo
- Publication number
- CN109293733A CN109293733A CN201811346576.8A CN201811346576A CN109293733A CN 109293733 A CN109293733 A CN 109293733A CN 201811346576 A CN201811346576 A CN 201811346576A CN 109293733 A CN109293733 A CN 109293733A
- Authority
- CN
- China
- Prior art keywords
- holoprotein
- tobacco
- pbs
- tobacco leaves
- extracting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application belongs to tobacco metabolin technical field, and in particular to a kind of extracting method patent application matters suitable for seedling stage fresh tobacco leaves holoprotein.Method is applicable in for seedling stage fresh tobacco leaves, is specifically included: preparing extracting solution (using lysate RIPA, buffer NEB or PBS), tobacco leaf pretreatment, solwution method are extracted.Preliminary Determination the result shows that, after associated extraction process parameter optimizing, the protein extraction amount of extracting method provided herein is although medium, but impurity is few, protein extraction effect is more preferable, and since associated extraction method is simple and convenient, time-consuming shorter (only 1.5 h or so), so that the application has preferable scientific research value, technical foundation can be established for the analysis of the GAP-associated protein GAPs such as the general immunoblotting of target protein, co-immunoprecipitation and research.
Description
Technical field
The application belongs to tobacco metabolin technical field, and in particular to one kind is suitable for seedling stage fresh tobacco leaves holoprotein
Extracting method patent application matters.
Background technique
For tobacco as a kind of more special industrial crops, yield and quality is directly related to the strong of Chinese national economy
Kang Fazhan.In order to study tobacco gene function, new breeding material is formulated, gene expression product protein is furtherd investigate
It is obviously had a very important significance with analysis.
In protein research, the preparation of protein sample is starting and the basis of analysis, therefore protein extraction quality and effect
Subsequent research and analyse is had a major impact.And since tobacco leaf contains thicker cell wall, and mentioning to protein tobacco
It takes and increases certain difficulty.In the prior art, there are mainly two types of the extracting methods of vegetable protein: solwution method and the precipitation method.Its
The middle precipitation method will lead to part protease structure and change and lose activity, therefore is heavy because using denaturant in its extraction process
Shallow lake method is not appropriate for the extraction of tobacco leaf holoprotein.And when solution application method extracts, due to different biologies and not
It is different with the protein characteristic of growth period, therefore selected extracting solution and extraction process are also tended to lack and can be borrowed when specific extraction
Mirror property, is often both needed to redesign.And for tobacco quality analysis and rearing new variety, it establishes a kind of for fresh tobacco
Blade, simple and quick efficient holoprotein extraction method all has the research of tobacco functional gene and the initiative of tobacco new material
There is highly important application value.
Summary of the invention
The application is designed to provide a kind of extracting method suitable for seedling stage fresh tobacco leaves holoprotein, to be cigarette
Certain methodology basis is established in careless growth metabolism research.
Details are as follows for the technical solution that the application is taken.
A kind of extracting method suitable for fresh tobacco leaves holoprotein, this method are applicable in for seedling stage fresh tobacco leaves, tool
Body includes the following steps:
(1) extracting solution is prepared, the PBS of lysate RIPA, NEB or difference pH specifically can be used；Formula composition are as follows:
Lysate RIPA:Tris-Cl(pH 8.0), 50 mM, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%,
PMSF,1 mM；
Buffer NEB:Hepes(pH 7.5), 20mM, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
The PBS:NaCl of pH 8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1
mM；
The PBS:NaCl of pH7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH 6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1
mM；
(2) tobacco leaf pre-processes
Tobacco K326(or NC82 will be cultivated) fresh tobacco leaves of 8-12 leaf phase, after liquid nitrogen flash freezer, pulverize.
(3) solwution method is extracted
It pulverizes the extracting solution (lysate) for being added in tobacco sample and being prepared in step (1) in step (2), after concussion mixes,
It stands on ice and is no less than 1h；
In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution (lysate) dosage is 500 μ l-1mL；
After the completion of standing, 5000 ~ 15000rpm centrifugation be no less than 20min(centrifugal speed specifically be, for example, 5000 rpm,
9000rpm or 13000rpm etc.), retain supernatant, as containing the extracting solution of holoprotein；
Further, protein concentration in supernatant can be detected using BCA method.
In the prior art, when carrying out analysis measurement for holoprotein, since extracted impurity is more, thus for albumen
Accurate Determining causes larger impact.In the application, part has been done for holoprotein extracting mode and has been optimized, to go to clean as far as possible
Matter influences, to improve purity of protein.
Preliminary Determination the result shows that, after associated extraction process parameter optimizing, the egg of extracting method provided herein
White extracted amount is although medium, but impurity is few, and protein extraction effect is more preferable, and since associated extraction method is simple and convenient, time-consuming
Shorter (only 1.5 h or so) so that the application have preferable scientific research value, can for the general immunoblotting of target protein,
Technical foundation is established in the analysis of the GAP-associated protein GAPs such as co-immunoprecipitation and research.
Detailed description of the invention
The holoprotein that Fig. 1 difference extracting solution, different rotating speeds extract；
Fig. 2 difference extracts the holoprotein concentration that liquid proportional extracts；
The holoprotein concentration that Fig. 3 difference cultivar obtains；
In each figure: K326, NEB, RIPA respectively indicate corresponding lysate, and P6, P7, P8 respectively indicate pH=6.0,7.2,8.0
PBS, it is 5000,13000 that number 5,13, which respectively indicates centrifugal speed, and 1:5,1:10 respectively indicate extraction liquid proportional.
Specific embodiment
Explanation is further explained to the application below with reference to embodiment, before introducing specific embodiment, with regard to following realities
It applies the backgrounds such as part Experiment material, experimental method involved in example and briefly introduces and be described as follows.
Experimental material:
Tobacco K326 and NC82 are cultivated, laboratory culture takes fresh tobacco leaves to the 8-12 leaf phase.
Experimental method:
When BCA method detects protein concentration, with specific reference to following operating procedure:
(1) supernatant is taken, dilutes 10 times；According to sample size, add 1 volume BCA reagent B(50:1 by 50 volume BCA reagent As) match
BCA working solution processed, mixes well；
(2) blank well, standard sample wells and sample well, each sample is arranged to repeat three times, 20 μ l protein extracts of every empty addition,
200 μ L BCA working solutions；
(3) ELISA Plate is put and vibrates 30sec on the oscillator, 37 DEG C are placed 30 minutes, and then microplate reader detects at 562nm,
With protein content (μ g) for abscissa, light absorption value is ordinate, draws standard curve；
According to the light absorption value of institute's sample, establishing criteria curve obtains corresponding protein content (μ g), total divided by sample diluting liquid
Volume (20 μ L) is sample actual concentrations (unit: μ g/ μ L) multiplied by sample extension rate.
Embodiment
The extracting method for improving holoprotein in tobacco leaf provided herein, is extracted for fresh tobacco leaves, specific steps letter
It is described below.
(1) extracting solution is prepared, lysate RIPA, NEB or PBS specifically can be used；Formula composition are as follows:
Lysate RIPA:Tris-Cl(pH 8.0), 50 mM, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%,
PMSF,1 mM；
Buffer NEB:Hepes(pH 7.5), 20mM, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
The PBS:NaCl of pH 8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1
mM；
The PBS:NaCl of pH7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH 6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1
mM；
(2) tobacco leaf pre-processes
Tobacco K326 and NC82 are cultivated, laboratory culture to 8-12 leaf phase acquires fresh tobacco leaves.After liquid nitrogen flash freezer, it is ground into
Powder.
(3) solwution method is extracted
It pulverizes the extracting solution (lysate) for being added in tobacco sample and being prepared in step (1) in step (2), after concussion mixes,
It stands on ice and is no less than 1h；
In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution (lysate) dosage is that 500 μ l-1mL(are specially
500 μ l or 1mL)；
After the completion of standing, 5000 ~ 15000rpm centrifugation be no less than 20min(centrifugal speed specifically be, for example, 5000 rpm,
9000rpm or 13000rpm etc.), retain supernatant, as containing the extracting solution of holoprotein；
Further, protein concentration in supernatant is detected using BCA method.
To complete in supernatant after the PBS of different extracting solutions, difference pH, different centrifugal speeds and different extraction liquid proportional processing
Protein concentration is detected, as a result as shown in Figure 1, Figure 2, Figure 3 shows respectively.Analysis is high it can be seen that for centrifugal speed parameter
Being obtained in the case of holoprotein concentration is obviously higher namely high speed centrifugation under fast parameter of noncentricity (13000 rpm) preferably to remove
Impurity effect；But the PBS of the PBS and pH 6.0 of pH 8.0 effect under the conditions of low-speed centrifugal is then more apparent due to high speed centrifugation,
According to the analysis, partial impurities contained therein affect measurement result in the case of low-speed centrifugal；For extracting liquid proportional, 1:10
The total protein content that ratio obtains is more；NC82 is also preferable using the protein concentration that the same terms obtain.
Generally, for holoprotein extraction, it will be apparent that, using lysate RIPA, high speed centrifugation (13000 rpm), 1:
10 extract liquid proportionals, and treated that protein content measurement result is substantially higher in other extracting solutions as a result, this is mainly due at cracking
After reason, the albumen contained by cell interior has obtained further release.
Claims (4)
1. a kind of extracting method suitable for fresh tobacco leaves holoprotein, which is characterized in that this method is directed to seedling stage fresh cigarette
Leaf is applicable in, and is specifically comprised the following steps:
(1) extracting solution is prepared, the specific PBS for using lysate RIPA, NEB or difference pH；Formula composition are as follows:
Tris-Cl, 50 mM of lysate RIPA: pH 8.0, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%,
PMSF,1 mM；
Hepes, 20mM of buffer NEB:pH 7.5, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
(2) tobacco leaf pre-processes
By the fresh tobacco leaves of tobacco 8-12 leaf phase, it is crushed into powder spare；
(3) solwution method is extracted
It is stood on ice after concussion mixes in step (2) extracting solution for being added in tobacco sample and being prepared in step (1) of pulverizing
No less than 1h；In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution dosage is 500 μ l-1mL；
After the completion of standing, 5000 ~ 15000rpm centrifugation is no less than 20min, retains supernatant, as containing the extraction of holoprotein
Liquid.
2. being suitable for the extracting method of fresh tobacco leaves holoprotein as described in claim 1, which is characterized in that in step (2),
The tobacco is K326 NC82 kind.
3. being suitable for the extracting method of fresh tobacco leaves holoprotein as claimed in claim 2, which is characterized in that in step (1)
PH=8.0 of PBS, 7.2 or 6.0, concrete composition are as follows:
The PBS:NaCl of pH=8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1 mM；
The PBS:NaCl of pH=7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH=6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1 mM.
4. being suitable for the extracting method of fresh tobacco leaves holoprotein as claimed in claim 3, which is characterized in that in step (1),
Using lysate RIPA；In step (3), extracting solution dosage is 1mL, and 13000 rpm are centrifuged 20min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811346576.8A CN109293733A (en) | 2018-11-13 | 2018-11-13 | A kind of extracting method suitable for fresh tobacco leaves holoprotein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811346576.8A CN109293733A (en) | 2018-11-13 | 2018-11-13 | A kind of extracting method suitable for fresh tobacco leaves holoprotein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109293733A true CN109293733A (en) | 2019-02-01 |
Family
ID=65146303
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811346576.8A Pending CN109293733A (en) | 2018-11-13 | 2018-11-13 | A kind of extracting method suitable for fresh tobacco leaves holoprotein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109293733A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305184A (en) * | 2019-07-15 | 2019-10-08 | 武汉轻工大学 | A kind of buffer and its application for co-immunoprecipitation |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870998A (en) * | 2010-07-13 | 2010-10-27 | 四川农业大学 | Method for detecting activity of plant superoxide dismutase, catalase and peroxidase |
| US20110257369A1 (en) * | 2008-10-17 | 2011-10-20 | University Of Maryland | Methods for removing nicotine and other alkaloids from soluble leaf proteins in solanaceous and other plant species |
| WO2012088295A1 (en) * | 2010-12-21 | 2012-06-28 | The Chinese University Of Hong Kong | A cost-effictive method for expression and purification of recombinant proteins in plants |
| CN102816758A (en) * | 2011-06-10 | 2012-12-12 | 清华大学 | Method for efficiently expressing foreign protein |
| CN103145817A (en) * | 2013-02-28 | 2013-06-12 | 华南农业大学 | Sickle-alfalfa chloroplast cold-response protein (MfcpCOR14) and coding gene and application thereof |
| CN104928317A (en) * | 2015-06-09 | 2015-09-23 | 昆明理工大学 | Plant expression vector with nicotiana tabacum gibberellins synthesis transcription regulation factor gene, and application of plant expression vector |
| CN105603047A (en) * | 2015-12-31 | 2016-05-25 | 中国烟草总公司郑州烟草研究院 | GSTs activity analysis method suitable for plant holoprotein extracting solution |
| CN107699579A (en) * | 2017-11-03 | 2018-02-16 | 南京农业大学 | A kind of gene for improving disease resistance of plant and its application |
-
2018
- 2018-11-13 CN CN201811346576.8A patent/CN109293733A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110257369A1 (en) * | 2008-10-17 | 2011-10-20 | University Of Maryland | Methods for removing nicotine and other alkaloids from soluble leaf proteins in solanaceous and other plant species |
| CN101870998A (en) * | 2010-07-13 | 2010-10-27 | 四川农业大学 | Method for detecting activity of plant superoxide dismutase, catalase and peroxidase |
| WO2012088295A1 (en) * | 2010-12-21 | 2012-06-28 | The Chinese University Of Hong Kong | A cost-effictive method for expression and purification of recombinant proteins in plants |
| CN102816758A (en) * | 2011-06-10 | 2012-12-12 | 清华大学 | Method for efficiently expressing foreign protein |
| CN103145817A (en) * | 2013-02-28 | 2013-06-12 | 华南农业大学 | Sickle-alfalfa chloroplast cold-response protein (MfcpCOR14) and coding gene and application thereof |
| CN104928317A (en) * | 2015-06-09 | 2015-09-23 | 昆明理工大学 | Plant expression vector with nicotiana tabacum gibberellins synthesis transcription regulation factor gene, and application of plant expression vector |
| CN105603047A (en) * | 2015-12-31 | 2016-05-25 | 中国烟草总公司郑州烟草研究院 | GSTs activity analysis method suitable for plant holoprotein extracting solution |
| CN107699579A (en) * | 2017-11-03 | 2018-02-16 | 南京农业大学 | A kind of gene for improving disease resistance of plant and its application |
Non-Patent Citations (3)
| Title |
|---|
| 尤垂淮等: "烟草叶片蛋白双向电泳技术体系的建立", 《中国烟草学报》 * |
| 董二慧等: "响应曲面法优化低次烟叶可溶性蛋白提取工艺", 《江苏农业科学》 * |
| 陈德鑫等: "烟草NteIF2α的原核表达、纯化及多克隆抗体制备和应用", 《农业生物技术学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305184A (en) * | 2019-07-15 | 2019-10-08 | 武汉轻工大学 | A kind of buffer and its application for co-immunoprecipitation |
| CN110305184B (en) * | 2019-07-15 | 2021-04-27 | 武汉轻工大学 | Buffer solution for co-immunoprecipitation and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| González-Neves et al. | Effect of cold pre-fermentative maceration on the color and composition of young red wines cv. Tannat | |
| CN105699142B (en) | Paraffin section preparation method for the close hard vegetable material of quality | |
| Sîrbu et al. | Physico-chemical and antioxidant properties of new sweet cherry cultivars from Iaşi, Romania | |
| CN101180310A (en) | Extraction of proteins from formalin-fixed tissue | |
| CN111139214B (en) | Liquid for extracting cell nucleus and application thereof | |
| CN103897018A (en) | Method for extracting total protein from soybean seeds and dedicated reagents for the method | |
| US20230009697A1 (en) | Method for identifying origin of chrysanthemi flos | |
| CN109293733A (en) | A kind of extracting method suitable for fresh tobacco leaves holoprotein | |
| CN104531679A (en) | Method for extracting DNA from dry apricot leaf | |
| KR101277749B1 (en) | Analysis of the purity of honey, using pollen mixed in honey | |
| CN109557228A (en) | A method of identifying bird's nest and its adulterant using signature peptide fragment | |
| WO2005112666A1 (en) | Method for extracting active components of ginseng by mechanical means | |
| CN108956679B (en) | Method for screening spruce embryo germination related marker metabolites based on NMR technology | |
| Stern et al. | Some chemical properties of isolated pea nucleoli | |
| CN110146431A (en) | A kind of preparation method and cell lysis buffer solution of the flow cytometry sample suitable for Calanthe plant | |
| CN104359738A (en) | Dry plant tissue treatment method applied to flow cytometry | |
| CN109321550A (en) | The extracting method of amylase enzyme activity in a kind of raising leaf tobacco extract | |
| CN104478987B (en) | A kind of special extract and extracting method of oil palm leaves total protein matter | |
| CN109321544A (en) | The extracting method of putrescine N-methyltransferase in a kind of raising leaf tobacco extract | |
| CN112321671A (en) | Method for extracting vegetable protein of rubber trees and hippocampus by phenol extraction method | |
| CN109321536A (en) | The extracting method of oxidizing ferment enzyme activity in a kind of raising leaf tobacco extract | |
| CN113740470A (en) | Method for judging whether loquat honey is mature or not and application | |
| CN111718394A (en) | Method for extracting sugarcane tissue denatured protein based on BPP method | |
| CN114002363A (en) | Application of phenyllactic acid as characteristic marker of Neliger Xinjiang black bee honey | |
| CN109619659B (en) | Application of tobacco nucleic acid extract in tobacco |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190201 |
|
| RJ01 | Rejection of invention patent application after publication |