CN103130866B - Plant plasma membrane protein redissolution method and application in two-dimensional fluorescent difference gel electrophoresis thereof - Google Patents
Plant plasma membrane protein redissolution method and application in two-dimensional fluorescent difference gel electrophoresis thereof Download PDFInfo
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Abstract
The invention discloses a plant plasma membrane protein redissolution method and application in two-dimensional fluorescent difference gel electrophoresis thereof. The plant plasma membrane protein redissolution method comprises the following steps: plant plasma membrane protein is added to a lysis solution, ultrasonic processing is conducted in an ice bath, then the plant plasma membrane protein is dissolved by eddy mixing, and at last supernatant fluid is collected in a centrifugal mode, namely a plant plasma membrane protein solution is obtained. The lysis solution is obtained by that N - dodecyl - beta - D - malt sugar is added to a fluorescent difference gel electrophoresis (DIGE) lysis buffer solution, and the concentration of the DIGE lysis buffer solution is set to be 0.5-2 g / 100 ML. The plant plasma membrane protein is low in abundance degree and high in hydrophobic property, certain difficulty exists in the extraction and the redissolution, and thus great challenge is brought to the research of plasma membrane proteomics. According to the plant plasma membrane protein redissolution method, the extraction efficiency of the plant plasma membrane protein is increased by nearly two times, the electrophoresis result of good repeatability and high resolution ration can be obtained after the 2-DDIGE is conducted, and more protein points are displayed compared with an existing method.
Description
Technical field
The present invention relates to a kind of method that plasmalemma of plant albumen dissolves again and the application in two-way fluorescence difference gel electrophoresis thereof.
Background technology
Plant plasma membrane serves is the barrier of cell and external environment, also be the key position that vegetable cell carries out the important vital movements such as exchange of substance, energy transformation and information transmission, in moisture, mineral ion, the transport of organic molecule cross-film, in the processes such as impression and transmission stress signal, play an important role.The main executive of plasma membrane function is plasmalemma protein, but plasmalemma of plant albumen abundance is low, hydrophobicity is strong, and there is the posttranslational modification such as glycosylation, phosphorylation in most of plasmalemma protein, cause the microheterogeneity of membranin, therefore how separation and purification and identification of cell membranin is the difficult point of plasmalemma of plant research.
The isolation identification of plant cytoplasm membranin mainly comprises method based on gel (gel-based) and the method based on gel (gel-free) not, the research of most of plasmalemma proteins adopts the not method based on gel, as membranin isolation identification or quantivative approach based on shotgun and 1-DE-LC-MS/MS, this method level of automation is high, flux is high, can successfully identify the stronger membranin of hydrophobicity, but the poor repeatability of result, cost is higher, the complexity of data is higher, and high-abundance proteins has been interfered the qualification of low-abundance protein, bring more difficulty to research.Although having report points out to be not suitable for based on the bidirectional electrophoresis technique of gel the plasmalemma protein of identifying that abundance is lower, hydrophobicity is strong, but this method has high-throughput, the feature such as directly perceived, is current only gordian technique that can simultaneously separate the protein mixture of large amount of complex.There is scientist to pass through the more not method based on gel and the quality of the method based on gel in the research of soybean leaves plasmalemma protein, finding that the experimental result of gel-free can not substitute the experimental result of gel-based completely, is good complementation between these two kinds of experimental techniques.
At present, more existing experiments that adopt bidirectional electrophoresis technique to analyze plasmalemma of plant albumen, but exist more problem: the extraction efficiency of plasmalemma of plant albumen is low, and the protein content that bidirectional electrophoresis technique needs is larger; Plasmalemma protein hydrophobicity is stronger, and indissoluble solution, in the plasmalemma protein glue more difficult to get access of indissoluble, has affected the isolation identification in later stage.The people such as Komatsu adopt the method for TCA/ acetone to precipitate the plasmalemma protein of soybean leaves, and it is carried out to Two-dimensional Electrophoresis Analysis, but its electrophoresis result is not very desirable, and laterally hangover is comparatively serious.The people such as Huang are by adding the solubilizing agent of plasmalemma protein to dissolve more plasmalemma protein, but the same laterally hangover of its electrophoresis result is comparatively serious, and resolving power is lower.Therefore, improve plasmalemma protein extraction efficiency, optimization plasmalemma protein again dissolving method is the difficult point of plasmalemma of plant protein science research.
In recent years, new fluorescent mark technology sequential use is in the research of two-dimensional electrophoresis.2-D DIGE (two-dimensional differential gel electrophoresis, two-way fluorescence difference gel electrophoresis) be a kind of emerging fluorescent mark quantitative proteomics technology, it has higher dynamics range and susceptibility than classical bidirectional electrophoresis technique, the sensitivity of qualification has reached pg level, can effectively identify low-abundance membranin, and in experiment, only need the albumen of 50 μ g, saved the consumption of plasmalemma protein in two-dimensional electrophoresis process, the appearance of this technology has advanced the plasmalemma protein group research based on gel greatly.At present, be applicable to the plasmalemma of plant protein extraction of this technology and again dissolving method not yet report, therefore, be necessary very much to set up a kind of plasmalemma of plant protein extraction of the 2-D of being applicable to DIGE technology and dissolving method again.
The research of plasmalemma of plant albumen focuses mostly in glycophyte (as paddy rice, soybean isotype plant), and halophytes is the important monoid in plant, plasma membrane plays an important role in its salt adaptive process, but less about the research of halophytes plasmalemma protein.Therefore, set up a set of plasmalemma of plant protein extraction and separation method that had not only been applicable to glycophyte but also had been applicable to halophytes, lay the foundation and be necessary for carrying out from now on the research of plasmalemma of plant albumen.
Summary of the invention
The object of this invention is to provide a kind of method that plasmalemma of plant albumen dissolves again and the application in two-way fluorescence difference gel electrophoresis thereof.
The method that plasmalemma of plant albumen provided by the invention dissolves again, comprise the steps: plasmalemma of plant albumen to add in lysate, supersound process in ice bath, then whirlpool is mixed and is made described plasmalemma of plant protein dissolution, last centrifugal collection supernatant liquor, is plasmalemma protein solution;
Described lysate adds N-dodecyl-β-D-Maltose glycosides to obtain in DIGE lysis buffer, and the concentration of described N-dodecyl-β-D-Maltose glycosides in described lysate is that 0.5-2g/100mL(is as 0.5-1g/100mL, 1-2g/100mL, 0.5g/100mL or 2g/100mL).The concentration of described N-dodecyl-β-D-Maltose glycosides in described lysate specifically can be 1g/100mL.
The parameter of described supersound process specifically can be: ultrasonic power 200W, and treatment time 30s, every work 3s stops 10s.
The mixed time of described whirlpool specifically can be 3h above (as 3h).
Described centrifugal parameter specifically can be: room temperature, the centrifugal 30min of 20000g.
Described DIGE lysis buffer is by solute and solvent composition, described solvent is 25mM Tris-HCl damping fluid (pH8.8), and described solute and the concentration in DIGE lysis buffer thereof are as follows: 7M urea, 2M thiocarbamide and 4g/100mL CHAPS(3-(3-(courage amido propyl group) dimethylamino) propanesulfonic acid).
The present invention also protects a kind of method of plasmalemma of plant protein solution for the preparation of carrying out 2-D DIGE, is to adopt the above method that plasmalemma of plant albumen is dissolved again, obtains plasmalemma of plant protein solution.
The preparation method of described plasmalemma of plant albumen can in turn include the following steps:
(1) in the suspension of plasmalemma of plant, add ice methyl alcohol, centrifugal after the mixed 4-10min in whirlpool;
(2) add chloroform, centrifugal after the mixed 4-10min in whirlpool;
(3) add ddH
2o, centrifugal after the mixed 4-10min in whirlpool, the uppermost one deck of reject;
(4) add ice methyl alcohol, centrifugal after the mixed 4-10min in whirlpool, collecting precipitation;
(5) by ice methanol wash precipitation, obtain plasmalemma of plant albumen.
In described step (1), described centrifugal can be instantaneous centrifugal.In described step (1), described whirlpool is mixed can at room temperature carry out, and does time and can be 5min in described whirlpool.In described step (2), described centrifugal can be instantaneous centrifugal.In described step (2), described whirlpool is mixed can at room temperature carry out, and does time and can be 5min in described whirlpool.In described step (3), described centrifugal parameter can be: room temperature, the centrifugal 1min of 13000g.In described step (3), described whirlpool is mixed can at room temperature carry out, and does time and can be 5min in described whirlpool.In described step (4), described centrifugal parameter can be: 4 DEG C, the centrifugal 5min of 13000g.In described step (4), described whirlpool is mixed can at room temperature carry out, and does time and can be 5min in described whirlpool.In the time that the volume of described plasmalemma of plant suspension is 100 μ l, in step (1), the add-on of ice methyl alcohol can be 400 μ l, and in step (2), the add-on of chloroform can be 150-250 μ l, ddH in step (3)
2the add-on of O can be 300 μ l, and in step (4), the add-on of ice methyl alcohol can be 300 μ l.
Described plasmalemma of plant suspension is specially plasmalemma of plant is suspended in to the suspension that diluent obtains.
Described plasmalemma of plant is specially the plasma membrane that extraction obtains from freezing plant sample.Described freezing plant sample specifically can be the powder that vegetable material (as the root system of the over-ground part of whole plant, plant or plant) is ground to form in liquid nitrogen.
The preparation method of described plasmalemma of plant suspension is specific as follows:
1, get the freezing plant sample of 40g, add the quiet 1h of carrying of lapping liquid, then grind and homogenate;
Lapping liquid (pH7.5) is by solute and solvent composition, and solvent is water, and solute and concentration thereof are as follows: 25mM HEPES, 0.33M sucrose, 10%(volume ratio) glycerine, 0.6g/100mL PVP, 5mM xitix, 5mM EDTA, 5mM DTT and 1mM PMSF.
The proportioning of lapping liquid and freezing sample is: 4mL:1g;
2, get the homogenate that step 1 obtains, filter and collect filtrate with double gauze, by 4 DEG C of filtrates, the centrifugal 15min of 10000g and collect supernatant liquor, by 4 DEG C of supernatant liquors, the centrifugal 1h of 80000g, collecting precipitation;
3, the precipitation obtaining by 9mL suspension suspension step 2, obtains total microcapsule suspension;
Suspension is by solute and solvent composition, and solvent is 5mM potassium phosphate buffer (pH7.8), and solute and concentration thereof are as follows: 0.33M sucrose, 3mM KCl, 1mM DTT and 1mM cocktail(proteinase inhibitor);
4, in the total microcapsule suspension obtaining in step 3, add two-phase liquid-1, the total microcapsule suspension of every 9g adds 27g two-phase liquid-1, mixes latter 4 DEG C, the centrifugal 10min of 1500g, and whole system is divided into two-phase;
Two-phase liquid-1 is by solute and solvent composition, solvent is 5mM potassium phosphate buffer (pH7.8), and solute and concentration thereof are as follows: 8.27%(mass ratio) Dextran T-500(dextran T-500), 8.27%(mass ratio) PEG3350(PEG3350), 0.33M sucrose and 3mM KCl;
5, get three parts of two-phase liquid-2, every part of 36g, 4 DEG C, the centrifugal 10min of 1500g, every individual system is divided into two-phase;
Two-phase liquid-2 are by solute and solvent composition, and solvent is 5mM potassium phosphate buffer (pH7.8), and solute and concentration thereof are as follows: 6.2%(mass ratio) Dextran T-500,6.2%(mass ratio) PEG3350,0.33M sucrose and 3mMKCl;
The lower phase liquid of a two-phase liquid-2 that obtain in the upper phase liquid 6, step 4 being obtained and step 5 mixes, 4 DEG C, the centrifugal 10min of 1500g, and system is divided into two-phase (two-phase system-A); The upper phase liquid of a two-phase liquid-2 that obtain in the lower phase liquid that step 4 is obtained and step 5 mixes, 4 DEG C, the centrifugal 10min of 1500g, and system is divided into two-phase (two-phase system-B).
7, the lower phase liquid of a two-phase liquid-2 that obtain in the upper phase liquid of two-phase system-A and step 5 is mixed, 4 DEG C, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-C); The upper phase liquid of a two-phase liquid-2 that obtain in the lower phase liquid that two-phase system-A is obtained and step 5 mixes, 4 DEG C, the centrifugal 10min of 1500g, and system is divided into two-phase (two-phase system-D); The lower phase liquid of a two-phase liquid-2 that obtain in the upper phase liquid of two-phase system-B and step 5 is mixed, 4 DEG C, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-E); The upper phase liquid of a two-phase liquid-2 that obtain in the lower phase liquid that two-phase system-B is obtained and step 5 mixes, 4 DEG C, the centrifugal 10min of 1500g, and system is divided into two-phase (two-phase system-F).
8, the upper phase liquid of the upper phase liquid of the upper phase liquid of the upper phase liquid of two-phase system-C, two-phase system-D, two-phase system-E and two-phase system-F is merged, with diluted to 10 times volume, then 4 DEG C, 12000g are centrifugal, collecting precipitation;
Diluent is by solute and solvent composition, and solvent is water, and solute and concentration thereof are as follows: 0.33M sucrose, 25mM HEPES and 1mM DTT;
9, the precipitation obtaining by the resuspended step 8 of 3mL diluent, then add Brij-58(tensio-active agent) and to make its concentration in system be 0.02g/100mL, place 10min on ice, then with diluted to 20 times volume, then 4 DEG C, the centrifugal 1h of 12000g, collecting precipitation, is used 500 μ l diluents resuspended, obtains plasmalemma of plant suspension.
Described plant can be monocotyledons or dicotyledons.Described plant can be glycophyte or halophytes.Described plant specifically can be salicornia europaeal or Arabidopis thaliana (Arabidopis thaliana as environmental in Colombia).
Arbitrary described method all can be used for 2-D DIGE above.
One of step of carrying out 2-D DIGE most critical is the preparation of protein sample, and plasmalemma of plant albumen abundance is low, and hydrophobicity is strong, and it to extract and to be dissolved into the difficult point in order studying again.The principal feature of the method that plasmalemma of plant albumen provided by the invention dissolves is again as follows: in (1) protein lysate, added gentle washing agent DM, had solublization, can increase the solubleness of plasmalemma of plant albumen; (2) after being added to lysate, plasmalemma of plant albumen carries out ultrasonication, and mixed to its continuous whirlpool with the mixed instrument in whirlpool afterwards, increased the dissolved efficiency of plasmalemma of plant albumen by physics means.The principal feature of the extracting method of plasmalemma of plant albumen provided by the invention is as follows: (1) has increased the addition of chloroform in leaching process, can be applicable to containing the more sample of phospholipid fraction; (2) proper extension the mixed required time in every step whirlpool, make Membrane protein extraction more abundant; (3) increase the step that washed with methanol precipitates, effectively removed contaminating impurity in albumen precipitation.
The extraction of general vegetable material plasma membrane adopts mill by fresh plant material homogenized, and the preservation of fresh plant material is the difficult point in actually operating.In the present invention, vegetable material liquid nitrogen flash freezer grind into powder are preserved.Extract when plasmalemma of plant, first, with the quiet vegetable material of carrying grind into powder of lapping liquid one hour, with lapping liquid, material is ground repeatedly afterwards, solved the preservation problem of fresh material.
The prerequisite of carrying out plasmalemma protein matter group is to obtain the plasma membrane composition that purity is higher, dextran/PVOH two phase partitions are separation and purification plasma membrane common methods, the selection of two-phase liquid concentration is different and different according to vegetable material, in the present invention, adopt 6.2% two-phase liquid concentration respectively salicornia europaeal root system, over-ground part and Arabidopis thaliana over-ground part to be carried out to purifying, and adopt 0.02%Brij-58 to make vesica upset, to reduce the pollution of plasma membrane attachment protein.Two-phase liquid concentration in the present invention and purification process can effectively be removed vacuole skin and these two kinds interior membrane components that easily pollute of endoplasmic reticulum in these three kinds of experiment materials, and present method has versatility.The acquisition of highly purified plasma membrane component is that late protein group analysis is laid a good foundation.
Utilizing comparison protein omics technology to analyze plasmalemma of plant albumen, is important laboratory facilities in plasmalemma of plant research.But the abundance of plasmalemma of plant albumen is low, and hydrophobicity is strong, its extraction and again dissolving exist certain difficulty, have brought great challenge to the research of plasmalemma protein matter group.Adopt plasmalemma of plant protein extraction provided by the invention and dissolving method again, the extraction efficiency of plasmalemma of plant albumen has increased nearly twice, carries out can obtaining after 2-D DIGE reproducible, and the electrophoresis result that resolving power is high shows more protein site than existing method.Therefore, present method can be used as a kind of current techique, is applied in the research of the plasmalemma of plant albumen based on 2-D DIGE technology from now on.
Brief description of the drawings
Fig. 1 is the immunoblotting collection of illustrative plates in embodiment 2, and a1, a2, a3 are the freezing sample of salicornia europaeal root system, and b1, b2, b3 are the freezing sample of salicornia europaeal over-ground part, and c1, c2, c3 are the freezing sample of Arabidopis thaliana over-ground part.
Fig. 2 is the 2-D DIGE collection of illustrative plates in embodiment 5, A is the 2-D DIGE collection of illustrative plates of the plasmalemma protein solution that obtains of the step 2 of embodiment 4, C is the enlarged view in a, b in A, c, d region, B is the 2-D DIGE collection of illustrative plates of the plasmalemma protein solution that obtains of the step 1 of embodiment 4, and D is the enlarged view in a, b in B, c, d region.
Fig. 3 is the 2-D DIGE collection of illustrative plates in embodiment 6, A is that the plasmalemma protein that the step 1 of embodiment 3 obtains carries out the 2-D DIGE collection of illustrative plates obtaining after embodiment 6, D is the enlarged view in a, b in A, c, d, e, f region, B is that the plasmalemma protein that the step 2 of embodiment 3 obtains carries out the 2-D DIGE collection of illustrative plates obtaining after embodiment 6, E is the enlarged view in a, b in B, c, d, e, f region, C is that the plasmalemma protein that the step 3 of embodiment 3 obtains carries out the 2-D DIGE collection of illustrative plates obtaining after embodiment 6, and F is the enlarged view in a, b in C, c, d, e, f region.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
The acquisition of the freezing sample of embodiment 1, vegetable material
Salicornia europaeal (Salicornia europaea L.) seed originates from Jiangsu Province of China Dafeng City's beach, can obtain from Jiangsu Jing Long marine industries Development Co., Ltd.Salicornia europaeal planting patterns is as follows: planting seed, to on the perlite of water-soaked, is put in to heliogreenhouse (the dark 8h of illumination 16h/; Day warm 25-30 DEG C, night temperature 18-20 DEG C; Relative humidity 60-80%) in, after it germinates, water weekly once with Hoagland nutritive medium, sow after 30 days and change into the Hoagland nutritive medium pouring that contains 200mM NaCl, after sowing 50 days, collect salicornia europaeal, get respectively its over-ground part and root system, grind into powder in liquid nitrogen, be the freezing sample of salicornia europaeal over-ground part and the freezing sample of salicornia europaeal root system, be placed in-80 DEG C of refrigerators and save backup.
The environmental Arabidopis thaliana of Colombia (Arabidopsis thaliana L.), seed is bought the stock in Arabidopsis Biological Resource Center (ABRC)).The environmental Arabidopis thaliana planting patterns of Colombia is as follows: be seeded in Nutrition Soil, cultivate (the dark 8h of illumination 16h/ in growth cabinet; 23 DEG C; Relative humidity 70%), watered once every three days, sow after seven weeks, get its over-ground part grind into powder in liquid nitrogen, be the freezing sample of Arabidopis thaliana over-ground part, be placed in-80 DEG C of refrigerators and save backup.
The extraction of embodiment 2, plasmalemma of plant component
The freezing sample of the freezing sample of the freezing sample of salicornia europaeal over-ground part, salicornia europaeal root system and Arabidopis thaliana over-ground part is proceeded as follows respectively:
One, the extraction of plasmalemma of plant component
1, get the freezing sample in 40g left and right, add the quiet 1h of carrying of lapping liquid, then grind and homogenate.
The solvent of lapping liquid (pH7.5) is water, contain following composition: 25mM HEPES(hydroxyethyl piperazine second thiosulfonic acid), 0.33M sucrose, 10%(volume ratio) glycerine, 0.6g/100mL PVP(polyvinylpyrrolidone), 5mM xitix, 5mM EDTA, 5mM DTT(dithiothreitol (DTT)) and 1mM PMSF(phenylmethylsulfonyl fluoride).
The proportioning of lapping liquid and freezing sample is: 4mL:1g.
2, get the homogenate that step 1 obtains, filter and collect filtrate with double gauze, by 4 DEG C of filtrates, the centrifugal 15min of 10000g and collect supernatant liquor, by 4 DEG C of supernatant liquors, the centrifugal 1h of 80000g, collecting precipitation.
3, the precipitation obtaining by 9mL suspension suspension step 2, obtains total microcapsule suspension.
Suspension is for containing 0.33M sucrose, 3mM KCl, 1mM DTT and 1mM cocktail(proteinase inhibitor) 5mM potassium phosphate buffer (pH7.8).
4, in the total microcapsule suspension obtaining in step 3, add two-phase liquid-1, the total microcapsule suspension of every 9g adds 27g two-phase liquid-1, and slowly turn upside down and mix approximately 100 times, 4 DEG C afterwards, the centrifugal 10min of 1500g, whole system is divided into obvious two-phase.
Two-phase liquid-1 is for containing 8.27%(mass ratio) Dextran T-500(dextran T-500), 8.27%(mass ratio) PEG3350(PEG3350), the 5mM potassium phosphate buffer (pH7.8) of 0.33M sucrose and 3mM KCl.
5, get three parts of two-phase liquid-2, every part of 36g, 4 DEG C, the centrifugal 10min of 1500g, every individual system is divided into two-phase.
Two-phase liquid-2 are for containing 6.2%(mass ratio) Dextran T-500,6.2%(mass ratio) the 5mM potassium phosphate buffer (pH7.8) of PEG3350,0.33M sucrose and 3mM KCl.
The lower phase liquid of a two-phase liquid-2 that obtain in the upper phase liquid 6, step 4 being obtained and step 5 mixes, 4 DEG C, the centrifugal 10min of 1500g, and system is divided into two-phase (two-phase system-A); The upper phase liquid of a two-phase liquid-2 that obtain in the lower phase liquid that step 4 is obtained and step 5 mixes, 4 DEG C, the centrifugal 10min of 1500g, and system is divided into two-phase (two-phase system-B).
7, the lower phase liquid of a two-phase liquid-2 that obtain in the upper phase liquid of two-phase system-A and step 5 is mixed, 4 DEG C, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-C); The upper phase liquid of a two-phase liquid-2 that obtain in the lower phase liquid that two-phase system-A is obtained and step 5 mixes, 4 DEG C, the centrifugal 10min of 1500g, and system is divided into two-phase (two-phase system-D); The lower phase liquid of a two-phase liquid-2 that obtain in the upper phase liquid of two-phase system-B and step 5 is mixed, 4 DEG C, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-E); The upper phase liquid of a two-phase liquid-2 that obtain in the lower phase liquid that two-phase system-B is obtained and step 5 mixes, 4 DEG C, the centrifugal 10min of 1500g, and system is divided into two-phase (two-phase system-F).
8, the upper phase liquid of the upper phase liquid of the upper phase liquid of the upper phase liquid of two-phase system-C, two-phase system-D, two-phase system-E and two-phase system-F is merged, with diluted to 10 times volume, then 4 DEG C, 12000g are centrifugal, collecting precipitation.
The solvent of diluent is water, contains following composition: 0.33M sucrose, 25mM HEPES and 1mM DTT.
9, the precipitation obtaining by the resuspended step 8 of 3mL diluent, then add Brij-58(tensio-active agent) and to make its concentration in system be 0.02g/100mL, place 10min(on ice so that vesicles overturns and washes away cytoplasmic pollutent), then with diluted to 20 times volume, then 4 DEG C, the centrifugal 1h of 12000g, collecting precipitation (being the plasmalemma of plant component that purifying obtains), used 500 μ l diluents resuspended, obtain plasmalemma of plant suspension, liquid nitrogen flash freezer is also stored in-80 DEG C of refrigerators.
Two, protein immunoblot analysis
Adopt Bradford colorimetry respectively the total microcapsule suspension (TM) in step 1 and plasma membrane suspension (PM) to be carried out to protein quantification.
Respectively the 9 plasma membrane suspension that obtain (containing 10 μ g plasmalemma proteins) of 3 of the step 1 total microcapsule suspension that obtain (containing the total microcapsule albumen of 10 μ g) and step 1 are carried out to 12.5%SDS-PAGE, then carry out immunoblotting assay.The primary antibodie adopting in immunoblotting assay is respectively for plasma membrane marker protein and (claims again H
+-ATPase albumen) primary antibodie (buying the company in Agrisera), for the primary antibodie (buying the company in Agrisera) of vacuole skin marker protein (claim not only V-ATPase albumen) with for the endoplasmic reticulum marker protein primary antibodie of (but also claiming SAR1 albumen) (buying the company in Agrisera), working concentration is 1:1000.Two goat anti-rabbit iggs that resist for horseradish peroxidase that adopt in immunoblotting assay, working concentration is 1:1000.
Fig. 1 is shown in by immunoblotting collection of illustrative plates.In total microcapsule suspension that the freezing sample of the freezing sample of salicornia europaeal over-ground part, the freezing sample of salicornia europaeal root system and Arabidopis thaliana over-ground part obtains, include plasma membrane, vacuole skin and endoplasmic reticulum.In the plasma membrane suspension that the freezing sample of the freezing sample of salicornia europaeal over-ground part, the freezing sample of salicornia europaeal root system and Arabidopis thaliana over-ground part obtains, all only has plasma membrane.
The extraction of embodiment 3, plasmalemma of plant albumen
One, the extracting method of plasmalemma of plant albumen provided by the invention
The plasmalemma of plant suspension that the freezing sample of the plasmalemma of plant suspension that the freezing sample of salicornia europaeal over-ground part is obtained, plasmalemma of plant suspension that the freezing sample of salicornia europaeal root system obtains and Arabidopis thaliana over-ground part obtains proceeds as follows respectively (the mixed instrument in whirlpool is purchased from its woods Bel instrument manufacturing model VORTEX-901 of company) successively:
1, in the plasmalemma of plant suspension obtaining in the step 1 of 100 μ l embodiment 2, add 400 μ l ice methyl alcohol, instantaneous centrifugal after the mixed 5min in whirlpool under room temperature.
2, add 200 μ l chloroforms, instantaneous centrifugal after the mixed 5min in whirlpool under room temperature.
3, add 300 μ l ddH
2o, the mixed 5min in whirlpool under room temperature, the then centrifugal 1min of 13000g under room temperature, now albumen is positioned at middle layer, the uppermost one deck of reject.
4, add 300 μ l ice methyl alcohol, the mixed 5min in whirlpool under room temperature, then 4 DEG C, the centrifugal 5min of 13000g, collecting precipitation.
5, will precipitate with ice methanol wash twice, the plasmalemma of plant albumen obtaining.
Two, TCA method is extracted plasmalemma of plant albumen
The plasmalemma of plant suspension that the freezing sample of the plasmalemma of plant suspension that the freezing sample of salicornia europaeal over-ground part is obtained, plasmalemma of plant suspension that the freezing sample of salicornia europaeal root system obtains and Arabidopis thaliana over-ground part obtains proceeds as follows respectively successively:
1, in the plasmalemma of plant suspension obtaining in the step 1 of embodiment 2, add 50%(volume ratio) trichloroacetic acid solution, the concentration that makes trichoroacetic acid(TCA) is 10%(volume ratio), 4 DEG C of standing 30min, then 4 DEG C, the centrifugal 10min of 15000g, collecting precipitation.
2, will precipitate with ice acetone cleaning twice, obtain plasmalemma of plant albumen.
Three, ammonium acetate method is extracted plasmalemma of plant albumen
The plasmalemma of plant suspension that the freezing sample of the plasmalemma of plant suspension that the freezing sample of salicornia europaeal over-ground part is obtained, plasmalemma of plant suspension that the freezing sample of salicornia europaeal root system obtains and Arabidopis thaliana over-ground part obtains proceeds as follows respectively successively:
1, the ammonium acetate solution (solvent is methyl alcohol, contains 100mM ammonium acetate) that adds 5 times of volumes in the plasmalemma of plant suspension obtaining in the step 1 of embodiment 2 precipitates and spends the night, then 4 DEG C, the centrifugal 15min of 20000g, collecting precipitation in-20 DEG C of refrigerators.
2, will precipitate by ice washed with methanol twice, obtain plasmalemma of plant albumen.
The plasmalemma protein pH obtaining due to TCA method obviously reduces, and needs to regulate with NaOH the pH of sample before DIGE mark, and easily makes to introduce error in experiment.And the plasmalemma protein pH that method provided by the invention and ammonium acetate method obtain variation is not obvious, without regulating sample pH value, can reduce the probability of introducing experimental error.
Dissolving again of embodiment 4, plasmalemma of plant albumen
One, method provided by the invention
The plasmalemma of plant albumen that the freezing sample of the plasmalemma of plant albumen that the freezing sample of salicornia europaeal over-ground part is obtained, plasmalemma of plant albumen that the freezing sample of salicornia europaeal root system obtains and Arabidopis thaliana over-ground part obtains proceeds as follows respectively successively:
The plasmalemma of plant albumen that the step 1 of embodiment 3 is extracted adds in the DIGE lysis buffer that contains 1g/100mL N--dodecyl-β-D-Maltose glycosides (DM), in ice bath, supersound process 30s(Ultrasonic Cell Disruptor originates from Shanghai Bi Lang company, when work, setting power is 200W, and every work 3s stops 10s; This step be must guard against protein solution excess Temperature in ultrasonic procedure and is destroyed lysis buffer composition), then be positioned in 4 DEG C of refrigerators, with in the mixed 3h(practical application in mixed instrument (being purchased from its woods Bel instrument manufacturing model VORTEX-5 of company) whirlpool, automatic whirlpool, 3h above all can) its plasmalemma protein is fully dissolved, then room temperature, the centrifugal 30min of 20000g, collect supernatant liquor, be plasmalemma of plant protein solution.
DIGE lysis buffer is for containing 7M urea, 2M thiocarbamide and 4g/100mL CHAPS(3-(3-(courage amido propyl group) dimethylamino) propanesulfonic acid) 25mM Tris-HCl damping fluid (pH8.8).
Two, existing method
The plasmalemma of plant albumen that the freezing sample of the plasmalemma of plant albumen that the freezing sample of salicornia europaeal over-ground part is obtained, plasmalemma of plant albumen that the freezing sample of salicornia europaeal root system obtains and Arabidopis thaliana over-ground part obtains proceeds as follows respectively successively:
The plasmalemma of plant albumen that the step 1 of embodiment 3 is extracted adds in DIGE lysis buffer, and vortex mixes, and then room temperature leaves standstill 2-3 hour, and then room temperature, the centrifugal 30min of 20000g collects supernatant liquor, is plasmalemma of plant protein solution.
The effect comparison of the dissolving method again of embodiment 5, plasmalemma of plant albumen of the present invention and the dissolving method again of existing plasmalemma of plant albumen
The plasmalemma of plant protein solution that the plasmalemma of plant protein solution that the step 1 of embodiment 4 is obtained and the step 2 of embodiment 4 obtain proceeds as follows respectively:
1, adopt NaOH to regulate the pH to 8.8 of plasmalemma protein solution, use Bradford colorimetric method for determining protein concentration.
The protein content that the salicornia europaeal root system of every gram of fresh weight carries out in plasmalemma protein solution that step 1, the step 1 of embodiment 3 and the step 1 of embodiment 4 of embodiment 1, embodiment 2 obtain is successively approximately 37.1 ± 5.8 μ g, the protein content that the salicornia europaeal root system of every gram of fresh weight carries out in plasmalemma protein solution that step 1, the step 1 of embodiment 3 and the step 2 of embodiment 4 of embodiment 1, embodiment 2 obtain is successively approximately 21.3 ± 4.2 μ g, adopt plasmalemma protein provided by the invention again dissolving method make albumen yield be increased to 1.74 times.
The protein content that the salicornia europaeal over-ground part of every gram of fresh weight carries out in plasmalemma protein solution that step 1, the step 1 of embodiment 3 and the step 1 of embodiment 4 of embodiment 1, embodiment 2 obtain is successively approximately 11.5 ± 1.6 μ g, the protein content that the salicornia europaeal over-ground part of every gram of fresh weight carries out in plasmalemma protein solution that step 1, the step 1 of embodiment 3 and the step 2 of embodiment 4 of embodiment 1, embodiment 2 obtain is successively approximately 6.4 ± 1.2 μ g, adopt plasmalemma protein provided by the invention again dissolving method make albumen yield be increased to 1.79 times.
The protein content that the Arabidopis thaliana over-ground part of every gram of fresh weight carries out in plasmalemma protein solution that step 1, the step 1 of embodiment 3 and the step 1 of embodiment 4 of embodiment 1, embodiment 2 obtain is successively approximately 5.7 ± 1.1 μ g, the protein content that the Arabidopis thaliana over-ground part of every gram of fresh weight carries out in plasmalemma protein solution that step 1, the step 1 of embodiment 3 and the step 2 of embodiment 4 of embodiment 1, embodiment 2 obtain is successively approximately 3.2 ± 0.8 μ g, adopt plasmalemma protein provided by the invention again dissolving method make albumen yield be increased to 1.78 times.
Above result shows, the dissolving method again of plasmalemma of plant albumen provided by the invention has improved the yield of plasmalemma of plant albumen greatly.
2, get the plasmalemma of plant protein solution containing 50 μ g albumen, add the DIGE dyestuff (Cy5) of 50pmol, more than being placed in the 2h of dark placement on ice, then add 1 μ l10mM lysine solution with termination reaction.
3, add isopyknic 2 × sample buffer (pH4-7, solvent is water, containing 7M urea, 2M thiocarbamide, 4%CHAPS, 2%DTT and 2%IPG), add afterwards protein lysate (pH4-7, solvent is water, containing 7M urea, 2M thiocarbamide, 4%CHAPS, 1%DTT and 1%IPG) to 450 μ l, after mixing, add in aquation groove, the adhesive tape that is 4-7 by 24cm pH gradient (GE Healthcare) was room temperature aquation 24 hours, afterwards with reference to manufacturer's specification sheets (2-DE Manual, GE Healthcare), adhesive tape is placed in to Ettan IPGphor electrophoresis system and carries out isoelectric focusing electrophoresis.
4,, after isoelectric focusing electrophoresis completes, adhesive tape is placed in to balance liquid (containing the 50mM Tris damping fluid of 6M urea, 30% glycerine, 2%SDS and 0.002% tetrabromophenol sulfonphthalein, pH8.8) and carries out balance.Balance carries out twice altogether, adds for the first time 1%DTT in balance liquid, adds for the second time 4% iodo-acid amide, each balance 15 minutes.
5, second adopt SDS-PAGE vertical electrophoresis to carry out (2-DE Manual, GE Healthcare) to electrophoresis.
6, second-phase electrophoresis uses Typhoon9400 (GE Healthcare) scanner scanning electrophoresis result figure after finishing, adjust sweep voltage, make the Max intensity of the protein site scanning between 60000 to 90000, the Max intensity difference between every glue figure is no more than 15%.The electrophoresis result that adopts DeCyder6.5 software to obtain scanning is analyzed.
Fig. 2 is shown in by the 2-D DIGE collection of illustrative plates that the freezing sample of salicornia europaeal root system obtains.In collection of illustrative plates, plasmalemma protein has obtained good separation, and the distribution range of protein site is wider, has substantially covered regional.Glue figure clean background, horizontal and vertical hangover is less.In the collection of illustrative plates of the plasmalemma protein solution that the method that compared with the conventional method, adopts invention to provide obtains, albumen is counted more.Choose four regions of different molecular weight and different pH scopes on glue figure and carried out local amplification ratio.Result shows, compared with the conventional method, (a, c), lower molecular weight district (b) in high molecular region, more protein site (black arrow instruction in figure) has all been isolated in pH meta-alkalescence region (d), and the more plasmalemma protein of cross-film comes across district of meta-alkalescence district (d) more.As we can see from the figure, the protein site of the plasmalemma protein solution that method provided by the invention obtains is more, and the Enrichment of Partial Protein point.In a word, adopt method provided by the invention to carry out plasmalemma protein and dissolve, plasmalemma protein extraction efficiency increases, and the membranin that separation obtains is counted also to be increased to some extent, for later stage isolation identification plasmalemma protein is laid a good foundation.
The analytical results of the 2-D DIGE collection of illustrative plates that the 2-D DIGE collection of illustrative plates that the 2-D DIGE collection of illustrative plates that the freezing sample of salicornia europaeal over-ground part obtains, the freezing sample of Arabidopis thaliana over-ground part obtain obtains with the freezing sample of salicornia europaeal root system is consistent.
The effect comparison of embodiment 6, plasmalemma of plant protein extracting method provided by the invention and existing plasmalemma of plant protein extracting method
1, the plasmalemma protein freezing sample of salicornia europaeal root system being obtained proceeds as follows successively:
The plasmalemma protein that the plasmalemma protein of the plasmalemma protein respectively step 1 of embodiment 3 being extracted, the extraction of the step 2 of embodiment 3 and the step 3 of embodiment 3 are extracted carries out the step 1 of embodiment 4, obtains three kinds of plasmalemma protein solution.
2, plasmalemma protein solution is carried out to 2-D DIGE according to the method for embodiment 5.
Fig. 3 is shown in by 2-D DIGE collection of illustrative plates.Find that the plasmalemma protein that three kinds of methods obtain carries out identical dissolving afterwards and put and exist roughly the same distribution again, the protein site that the plasmalemma protein that method provided by the invention obtains obtains after dissolving is again (2099) at most, the protein site that the plasmalemma protein that TCA method obtains obtains after dissolving is again 1652, and the protein site that the plasmalemma protein that ammonium acetate method obtains carries out obtaining after dissolving is 1856.
Every glue figure is divided into a-fLiu Ge district by molecular weight and pH, carries out amplification ratio.In a, c, d, e, f Wu Ge district, the protein site that the protein site that the plasmalemma protein that the protein site that the plasmalemma protein that method provided by the invention obtains obtains after dissolving again obviously obtains more than TCA method obtains after dissolving again, the obvious plasmalemma protein obtaining more than ammonium acetate method obtain after dissolving again.
By above-mentioned comparison, the protein site that adopts method extraction plasmalemma protein provided by the invention to obtain at most of region disconnectings is many compared with other two kinds of methods, the plasmalemma protein efficiency of extracting is higher, and in the time of DIGE mark, does not need to regulate protein lysate pH, can avoid experimental error.Meanwhile, the albumen species number that this method obtains is more compared with other two kinds of methods, is therefore more suitable for the 2-D DIGE of plasmalemma protein.
Dissolving again of embodiment 7, plasmalemma protein
1, dissolving again of plasmalemma protein
The plasmalemma of plant albumen that the freezing sample of salicornia europaeal root system is obtained proceeds as follows successively:
The plasmalemma of plant albumen that the step 1 of embodiment 3 is extracted adds in the DIGE lysis buffer that contains 0.5g/100mL N--dodecyl-β-D-Maltose glycosides (DM), in ice bath, supersound process 30s(Ultrasonic Cell Disruptor originates from Shanghai Bi Lang company, when work, setting power is 200W, and every work 3s stops 10s; This step be must guard against protein solution excess Temperature in ultrasonic procedure and is destroyed lysis buffer composition), then be positioned in 4 DEG C of refrigerators, with Hun Yi whirlpool, automatic whirlpool, mixed 3h fully dissolves its plasmalemma protein, then room temperature, the centrifugal 30min of 20000g, collect supernatant liquor, be plasmalemma of plant protein solution.
2, dissolving again of plasmalemma protein
The plasmalemma of plant albumen that the freezing sample of salicornia europaeal root system is obtained proceeds as follows successively:
The plasmalemma of plant albumen that the step 1 of embodiment 3 is extracted adds in the DIGE lysis buffer that contains 2g/100mL N-dodecyl-β-D-Maltose glycosides (DM), in ice bath, supersound process 30s(Ultrasonic Cell Disruptor originates from Shanghai Bi Lang company, when work, setting power is 200W, and every work 3s stops 10s; This step be must guard against protein solution excess Temperature in ultrasonic procedure and is destroyed lysis buffer composition), then be positioned in 4 DEG C of refrigerators, with in the mixed 3h(practical application in Hun Yi whirlpool, automatic whirlpool, 3h above all can) its plasmalemma protein is fully dissolved, then room temperature, the centrifugal 30min of 20000g, collect supernatant liquor, be plasmalemma of plant protein solution.
3, the plasmalemma of plant protein solution that plasmalemma of plant protein solution step 1 being obtained and step 2 obtain carries out respectively 2-D DIGE, and method is shown in embodiment 5, and collection of illustrative plates is all consistent with Fig. 2 B.
The extraction of embodiment 8, plasmalemma of plant albumen
1, the extraction of plasmalemma of plant albumen
The plasmalemma of plant suspension that the freezing sample of salicornia europaeal root system is obtained proceeds as follows successively:
(1) in the plasmalemma of plant suspension obtaining in the step 1 of 100 μ l embodiment 2, add 400 μ l ice methyl alcohol, instantaneous centrifugal after the mixed 4min in whirlpool under room temperature.
(2) add 150 μ l chloroforms, instantaneous centrifugal after the mixed 4min in whirlpool under room temperature.
(3) add 300 μ l ddH
2o, the mixed 4min in whirlpool under room temperature, the then centrifugal 1min of 13000g under room temperature, now albumen is positioned at middle layer, the uppermost one deck of reject.
(4) add 300 μ l ice methyl alcohol, the mixed 4min in whirlpool under room temperature, then 4 DEG C, the centrifugal 5min of 13000g,
Collecting precipitation.
(5) will precipitate with ice methanol wash twice, the plasmalemma of plant albumen obtaining.
2, the extraction of plasmalemma of plant albumen
The plasmalemma of plant suspension that the freezing sample of salicornia europaeal root system is obtained proceeds as follows successively:
(1) in the plasmalemma of plant suspension obtaining in the step 1 of 100 μ l embodiment 2, add 400 μ l ice methyl alcohol, instantaneous centrifugal after the mixed 10min in whirlpool under room temperature.
(2) add 250 μ l chloroforms, instantaneous centrifugal after the mixed 10min in whirlpool under room temperature.
(3) add 300 μ l ddH
2o, the mixed 10min in whirlpool under room temperature, the then centrifugal 1min of 13000g under room temperature, now albumen is positioned at middle layer, the uppermost one deck of reject.
(4) add 300 μ l ice methyl alcohol, the mixed 10min in whirlpool under room temperature, then 4 DEG C, the centrifugal 5min of 13000g, collecting precipitation.
(5) will precipitate with ice methanol wash twice, the plasmalemma of plant albumen obtaining.
3, the plasmalemma of plant albumen that the plasmalemma of plant albumen respectively step 1 being extracted and step 2 are extracted carries out the step 1 of embodiment 4, obtains two kind of plant plasmalemma protein solution.
4, plasmalemma of plant protein solution is carried out to 2-D DIGE according to the method for embodiment 5.
Collection of illustrative plates is all consistent with Fig. 3 A.
Claims (6)
1. for the preparation of the method for plasmalemma of plant protein solution of carrying out 2-D DIGE, be that the following method of plasmalemma of plant albumen is dissolved again, obtain plasmalemma of plant protein solution; The preparation method of described plasmalemma of plant albumen in turn includes the following steps:
(1) in plasmalemma of plant suspension, add ice methyl alcohol, centrifugal after the mixed 4-10min in whirlpool;
(2) add chloroform, centrifugal after the mixed 4-10min in whirlpool;
(3) add ddH
2o, centrifugal after the mixed 4-10min in whirlpool, the uppermost one deck of reject;
(4) add ice methyl alcohol, centrifugal after the mixed 4-10min in whirlpool, collecting precipitation;
(5) by ice methanol wash precipitation, obtain plasmalemma of plant albumen;
The method that described plasmalemma of plant albumen dissolves again, comprises the steps: plasmalemma of plant albumen to add in lysate, supersound process in ice bath, and then whirlpool is mixed makes described plasmalemma of plant protein dissolution, and last centrifugal collection supernatant liquor, is plasmalemma of plant protein solution; Described lysate adds N-dodecyl-β-D-Maltose glycosides to obtain in DIGE lysis buffer, and the concentration of described N-dodecyl-β-D-Maltose glycosides in described lysate is 0.5-2g/100mL.
2. the method for claim 1, is characterized in that: the concentration of described N-dodecyl-β-D-Maltose glycosides in described lysate is 1g/100mL.
3. method as claimed in claim 2, is characterized in that: described plant is monocotyledons or dicotyledons.
4. method as claimed in claim 3, is characterized in that: described plant is glycophyte or halophytes.
5. method as claimed in claim 4, is characterized in that: described plant is salicornia europaeal or Arabidopis thaliana.
6. the application of arbitrary described method in 2-D DIGE in claim 1 to 5.
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| CN102183398A (en) * | 2011-02-24 | 2011-09-14 | 中山大学 | Method for external specificity fluorescent staining of methane-oxidizing bacteria |
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| CN102183398A (en) * | 2011-02-24 | 2011-09-14 | 中山大学 | Method for external specificity fluorescent staining of methane-oxidizing bacteria |
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