CN103130866A - 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 method and the application in two-way fluorescence difference gel electrophoresis thereof that a kind of plasmalemma of plant albumen dissolves again.
Background technology
Plant plasma membrane serves is the barrier of cell and external environment, it is also 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 transportation of organic molecule cross-film, play an important role in the processes such as impression and transmission stress signal.The main executive of plasma membrane function is plasmalemma protein, yet plasmalemma of plant albumen abundance is low, hydrophobicity is strong, and there are the posttranslational modifications such as glycosylation, phosphorylation in most of plasmalemma protein, cause the microheterogeneity of membranin, therefore how separation and purification and identification of cell membranin, be the difficult point of plasmalemma of plant research.
the isolation identification of plant cytoplasm membranin mainly comprises based on the method for gel (gel-based) with not based on the method for gel (gel-free), the method based on gel is adopted not in the research of most of plasmalemma proteins, as membranin isolation identification or the 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 evaluation of low-abundance protein, bring more difficulty to research.Although being arranged, report points out that bidirectional electrophoresis technique based on gel is not suitable for the plasmalemma protein of identifying that abundance is lower, hydrophobicity is strong, but this method has high-throughput, the characteristics such as directly perceived, is the current only gordian technique that can separate simultaneously the protein mixture of large amount of complex.There is scientist to pass through more not based on the method for gel and the quality of method in the research of soybean leaves plasmalemma protein based on gel, the experimental result of finding gel-free can not substitute the experimental result of gel-based fully, is good complementation between these two kinds of experimental techniques.
At present, more existing experiments of adopting bidirectional electrophoresis technique that plasmalemma of plant albumen is analyzed, 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; The plasmalemma protein hydrophobicity is stronger, and the indissoluble solution in the plasmalemma protein of indissoluble glue more difficult to get access, 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 Two-dimensional Electrophoresis Analysis, but its electrophoresis result is not very desirable, and laterally hangover is comparatively serious.The solubilizing agent of the people such as Huang by adding plasmalemma protein is dissolving more plasmalemma protein, but the same laterally hangover of its electrophoresis result is comparatively serious, and resolving power is lower.Therefore, improve the plasmalemma protein extraction efficiency, optimization plasmalemma protein dissolving method again 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 the bidirectional electrophoresis technique of classics, the sensitivity of identifying has reached the pg level, can effectively identify low-abundance membranin, and only need the albumen of 50 μ g in experiment, saved the consumption of plasmalemma protein in the two-dimensional electrophoresis process, the appearance of this technology has advanced the plasmalemma protein group research based on gel greatly.At present, the plasmalemma of plant protein extraction that is applicable to this technology reaches dissolving method again and not yet reports, therefore, is necessary that very the plasmalemma of plant protein extraction of setting up a kind of 2-D of being applicable to DIGE technology reaches 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 plasmalemma of plant protein extraction and separation method that a cover not only had been applicable to glycophyte but also had been applicable to halophytes, be necessary for the research of carrying out from now on plasmalemma of plant albumen lays the foundation.
Summary of the invention
The purpose of this invention is to provide method and the application in two-way fluorescence difference gel electrophoresis thereof that a kind of plasmalemma of plant albumen dissolves again.
The method that plasmalemma of plant albumen provided by the invention dissolves again, comprise the steps: plasmalemma of plant albumen is added in lysate, supersound process in ice bath, then the 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 the DIGE lysis buffer, and the described N-dodecyl-β-concentration of D-Maltose glycosides in described lysate is 0.5-2g/100mL(such as 0.5-1g/100mL, 1-2g/100mL, 0.5g/100mL or 2g/100mL).The described N-dodecyl-β-concentration of 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 the 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 the 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 the plasmalemma of plant protein solution.
The preparation method of described plasmalemma of plant albumen can in turn include the following steps:
(1) add ice methyl alcohol in the suspension of plasmalemma of plant, 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) with 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 ℃, 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.When the volume of described plasmalemma of plant suspension was 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 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 over-ground part of whole plant, plant or the root system of 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, with 4 ℃ of filtrates, the centrifugal 15min of 10000g and collect supernatant liquor, with 4 ℃ of supernatant liquors, the centrifugal 1h of 80000g, collecting precipitation;
3, the precipitation that obtains with 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, add two-phase liquid-1 in total microcapsule suspension that step 3 obtains, the total microcapsule suspension of every 9g adds 27g two-phase liquid-1,4 ℃, the centrifugal 10min of 1500g after mixing, 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: the 8.27%(mass ratio) Dextran T-500(dextran T-500), the 8.27%(mass ratio) the PEG3350(PEG3350), 0.33M sucrose and 3mM KCl;
5, get three parts of two-phase liquid-2, every part of 36g, 4 ℃, 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: the 6.2%(mass ratio) Dextran T-500,6.2%(mass ratio) PEG3350,0.33M sucrose and 3mMKCl;
The lower phase liquid mixing of a two-phase liquid-2 that obtains in the upper phase liquid that 6, step 4 is obtained and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-A); The upper phase liquid mixing of a two-phase liquid-2 that obtains in the lower phase liquid that step 4 is obtained and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-B).
7, with the lower phase liquid mixing of a two-phase liquid-2 that obtains in the upper phase liquid of two-phase system-A and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-C); The upper phase liquid mixing of a two-phase liquid-2 that obtains in the lower phase liquid that two-phase system-A is obtained and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-D); With the lower phase liquid mixing of a two-phase liquid-2 that obtains in the upper phase liquid of two-phase system-B and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-E); The upper phase liquid mixing of a two-phase liquid-2 that obtains in the lower phase liquid that two-phase system-B is obtained and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-F).
8, the upper phase liquid of the upper phase liquid of two-phase system-C, two-phase system-D, the upper phase liquid of two-phase system-E and the upper liquid mutually of two-phase system-F are merged, with diluted to 10 times volume, then 4 ℃, 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 that obtains with the resuspended step 8 of 3mL diluent, then add the 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 ℃, the centrifugal 1h of 12000g, collecting precipitation, it is resuspended with 500 μ l diluents, obtain 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).
Above arbitrary described method all can be used for 2-D DIGE.
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 extracts and the difficult point that is dissolved into again in order to study.The principal feature of the method that plasmalemma of plant albumen provided by the invention dissolves again is as follows: added gentle washing agent DM in (1) protein lysate, had solublization, can increase the solubleness of plasmalemma of plant albumen; (2) after being added 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 the 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 the leaching process, can be applicable to contain the more sample of phosphatide component; (2) proper extension the mixed required time in per step whirlpool, make Membrane protein extraction more abundant; (3) increase the step of washed with methanol precipitation, effectively removed contaminating impurity in albumen precipitation.
The extraction of general vegetable material plasma membrane adopts mill with 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 and grind into powder are preserved.When extracting 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 higher plasma membrane composition of purity, dextran/PVOH two phase partitions are separation and purification plasma membrane common methods, the selective basis vegetable material of two-phase liquid concentration is different and different, in the present invention, the two-phase liquid concentration of employing 6.2% is carried out purifying to salicornia europaeal root system, over-ground part and Arabidopis thaliana over-ground part respectively, and adopt 0.02%Brij-58 to make the 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 the late protein group analysis is laid a good foundation.
Utilizing the comparison protein omics technology that plasmalemma of plant albumen is analyzed, is important laboratory facilities in plasmalemma of plant research.But the abundance of plasmalemma of plant albumen is low, and hydrophobicity is strong, and it extracts and dissolve and exists certain difficulty, has brought great challenge for the research of plasmalemma protein matter group.Adopt plasmalemma of plant protein extraction provided by the invention to reach dissolving method again, the extraction efficiency of plasmalemma of plant albumen has increased nearly twice, carries out accessing good reproducibility after 2-D DIGE, 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 plasmalemma of plant albumen research based on 2-D DIGE technology from now on.
Description of 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 zone, 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 zone.
Fig. 3 is the 2-D DIGE collection of illustrative plates in embodiment 6, A is that plasmalemma protein that the step 1 of embodiment 3 obtains carries out the 2-D DIGE collection of illustrative plates that obtains after embodiment 6, D is the enlarged view in a, b in A, c, d, e, f zone, B is that plasmalemma protein that the step 2 of embodiment 3 obtains carries out the 2-D DIGE collection of illustrative plates that obtains after embodiment 6, E is the enlarged view in a, b in B, c, d, e, f zone, C is that plasmalemma protein that the step 3 of embodiment 3 obtains carries out the 2-D DIGE collection of illustrative plates that obtains after embodiment 6, and F is the enlarged view in a, b in C, c, d, e, f zone.
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 repeated experiments three times, results averaged.
The acquisition of the freezing sample of embodiment 1, vegetable material
Salicornia europaeal (Salicornia europaea L.) seed originates from China Jiangsu Province Dafeng City's beach, can obtain from the Jiangsu grand marine industries of crystalline substance Development Co., Ltd.The salicornia europaeal planting patterns is as follows: planting seed to on the perlite of water-soaked, is put in heliogreenhouse (the dark 8h of illumination 16h/; Warm 25-30 ℃ of day, temperature 18-20 ℃ of night; Relative humidity 60-80%) in, after it germinates, water weekly once with the Hoagland nutritive medium, sow the Hoagland nutritive medium pouring that changes into after 30 days with containing 200mM NaCl, collect salicornia europaeal after sowing 50 days, 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 ℃ of refrigerators and save backup.
The environmental Arabidopis thaliana (Arabidopsis thaliana L.) of Colombia, 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 ℃; Relative humidity 70%), watered once every three days, after seven weeks of sowing, 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 ℃ of refrigerators and save backup.
The extraction of embodiment 2, plasmalemma of plant component
The freezing sample of salicornia europaeal over-ground part, the freezing sample of salicornia europaeal root system and the freezing sample of Arabidopis thaliana over-ground part are 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, with 4 ℃ of filtrates, the centrifugal 15min of 10000g and collect supernatant liquor, with 4 ℃ of supernatant liquors, the centrifugal 1h of 80000g, collecting precipitation.
3, the precipitation that obtains with 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, add two-phase liquid-1 in total microcapsule suspension that step 3 obtains, the total microcapsule suspension of every 9g adds 27g two-phase liquid-1, the mixing approximately 100 times of slowly turning upside down, and 4 ℃ afterwards, the centrifugal 10min of 1500g, whole system is divided into obvious two-phase.
Two-phase liquid-1 is for containing the 8.27%(mass ratio) Dextran T-500(dextran T-500), the 8.27%(mass ratio) the 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 ℃, the centrifugal 10min of 1500g, every individual system is divided into two-phase.
Two-phase liquid-2 are for containing the 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 mixing of a two-phase liquid-2 that obtains in the upper phase liquid that 6, step 4 is obtained and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-A); The upper phase liquid mixing of a two-phase liquid-2 that obtains in the lower phase liquid that step 4 is obtained and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-B).
7, with the lower phase liquid mixing of a two-phase liquid-2 that obtains in the upper phase liquid of two-phase system-A and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-C); The upper phase liquid mixing of a two-phase liquid-2 that obtains in the lower phase liquid that two-phase system-A is obtained and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-D); With the lower phase liquid mixing of a two-phase liquid-2 that obtains in the upper phase liquid of two-phase system-B and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-E); The upper phase liquid mixing of a two-phase liquid-2 that obtains in the lower phase liquid that two-phase system-B is obtained and step 5,4 ℃, the centrifugal 10min of 1500g, system is divided into two-phase (two-phase system-F).
8, the upper phase liquid of the upper phase liquid of two-phase system-C, two-phase system-D, the upper phase liquid of two-phase system-E and the upper liquid mutually of two-phase system-F are merged, with diluted to 10 times volume, then 4 ℃, 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 that obtains with the resuspended step 8 of 3mL diluent, then add the 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 ℃, the centrifugal 1h of 12000g, collecting precipitation (being the plasmalemma of plant component that purifying obtains), it is resuspended with 500 μ l diluents, obtain plasmalemma of plant suspension, liquid nitrogen flash freezer also is stored in-80 ℃ of refrigerators.
Two, protein immunoblot analysis
Adopt the Bradford colorimetry respectively the total microcapsule suspension (TM) in step 1 and plasma membrane suspension (PM) to be carried out protein quantification.
The 9 plasma membrane suspension that obtain (containing 10 μ g plasmalemma proteins) of the total microcapsule suspension that respectively 3 of step 1 is obtained (containing the 10 total microcapsule albumen of μ g) and step 1 carry out 12.5%SDS-PAGE, then carry out immunoblotting assay.The primary antibodie that adopts in immunoblotting assay is respectively for the 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.That adopts in immunoblotting assay two anti-is the goat anti-rabbit igg of horseradish peroxidase, and working concentration is 1:1000.
The immunoblotting collection of illustrative plates is seen Fig. 1.Include plasma membrane, vacuole skin and endoplasmic reticulum 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.All only has plasma membrane 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.
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, add 400 μ l ice methyl alcohol in the plasmalemma of plant suspension that the step 1 of 100 μ l embodiment 2 obtains, 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 centrifugal 1min of 13000g under room temperature then, this moment, albumen was positioned at the 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 ℃, the centrifugal 5min of 13000g, collecting precipitation.
5, will precipitate with ice methanol wash twice, the plasmalemma of plant albumen that obtains.
Two, the 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, add the 50%(volume ratio in the plasmalemma of plant suspension that the step 1 of embodiment 2 obtains) trichloroacetic acid solution, the concentration that makes trichoroacetic acid(TCA) is the 10%(volume ratio), 4 ℃ of standing 30min, then 4 ℃, the centrifugal 10min of 15000g, collecting precipitation.
2, will precipitate with ice acetone cleaning twice, obtain plasmalemma of plant albumen.
Three, the 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, add the ammonium acetate solution (solvent is methyl alcohol, contains the 100mM ammonium acetate) of 5 times of volumes in the plasmalemma of plant suspension that the step 1 of embodiment 2 obtains, precipitate in-20 ℃ of refrigerators and spend the night, then 4 ℃, the centrifugal 15min of 20000g, collecting precipitation.
2, will precipitate with ice washed with methanol twice, obtain plasmalemma of plant albumen.
The plasmalemma protein pH that obtains due to the TCA method obviously reduces, and needs to regulate with NaOH the pH of sample before the DIGE mark, introduces error in experiment and easily make.And the plasmalemma protein pH that method provided by the invention and ammonium acetate method obtain variation is not obvious, need not to regulate sample pH value, can reduce the probability of introducing experimental error.
The 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), supersound process 30s(Ultrasonic Cell Disruptor originates from Shanghai than bright company in ice bath, during work, setting power is 200W, and every work 3s stops 10s; This step must guard against protein solution excess Temperature in ultrasonic procedure and destroys the lysis buffer composition), then be positioned in 4 ℃ of refrigerators, 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, more than 3h all can) its plasmalemma protein is fully dissolved, then room temperature, the centrifugal 30min of 20000g, collect supernatant liquor, be the plasmalemma of plant protein solution.
The 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 the DIGE lysis buffer, the vortex mixing, and then the standing 2-3 of room temperature hour, then room temperature, the centrifugal 30min of 20000g, collected supernatant liquor, is the plasmalemma of plant protein solution.
The effect 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 relatively
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 fresh weight carries out in plasmalemma protein solution that the step 1 of the step 1 of step 1, embodiment 3 of embodiment 1, embodiment 2 and embodiment 4 obtains successively is approximately 37.1 ± 5.8 μ g, the protein content that the salicornia europaeal root system of every gram fresh weight carries out in plasmalemma protein solution that the step 2 of the step 1 of step 1, embodiment 3 of embodiment 1, embodiment 2 and embodiment 4 obtains successively is approximately 21.3 ± 4.2 μ g, namely adopt plasmalemma protein provided by the invention again dissolving method make the albumen yield be increased to 1.74 times.
The protein content that the salicornia europaeal over-ground part of every gram fresh weight carries out in plasmalemma protein solution that the step 1 of the step 1 of step 1, embodiment 3 of embodiment 1, embodiment 2 and embodiment 4 obtains successively is approximately 11.5 ± 1.6 μ g, the protein content that the salicornia europaeal over-ground part of every gram fresh weight carries out in plasmalemma protein solution that the step 2 of the step 1 of step 1, embodiment 3 of embodiment 1, embodiment 2 and embodiment 4 obtains successively is approximately 6.4 ± 1.2 μ g, namely adopt plasmalemma protein provided by the invention again dissolving method make the albumen yield be increased to 1.79 times.
The protein content that the Arabidopis thaliana over-ground part of every gram fresh weight carries out in plasmalemma protein solution that the step 1 of the step 1 of step 1, embodiment 3 of embodiment 1, embodiment 2 and embodiment 4 obtains successively is approximately 5.7 ± 1.1 μ g, the protein content that the Arabidopis thaliana over-ground part of every gram fresh weight carries out in plasmalemma protein solution that the step 2 of the step 1 of step 1, embodiment 3 of embodiment 1, embodiment 2 and embodiment 4 obtains successively is approximately 3.2 ± 0.8 μ g, namely adopt plasmalemma protein provided by the invention again dissolving method make the 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 that contains 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, contain 7M urea, the 2M thiocarbamide, 4%CHAPS, 2%DTT and 2%IPG), add afterwards protein lysate (pH4-7, solvent is water, contain 7M urea, the 2M thiocarbamide, 4%CHAPS, 1%DTT and 1%IPG) to 450 μ l, add after mixing in the aquation groove, be that the adhesive tape (GE Healthcare) of 4-7 was room temperature aquation 24 hours with 24cm pH gradient, afterwards with reference to manufacturer's specification sheets (2-DE Manual, GE Healthcare), adhesive tape is placed in Ettan IPGphor electrophoresis system carries out isoelectric focusing electrophoresis.
4, after isoelectric focusing electrophoresis is completed, adhesive tape is placed in balance liquid (contains the 50mM Tris damping fluid of 6M urea, 30% glycerine, 2%SDS and 0.002% tetrabromophenol sulfonphthalein, pH8.8) carry 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 the SDS-PAGE vertical electrophoresis to carry out (2-DE Manual, GE Healthcare) to electrophoresis.
6, use Typhoon9400 (GE Healthcare) scanner scanning electrophoresis result figure after the second-phase electrophoresis finishes, adjust sweep voltage, make the Max intensity of protein site of scanning between 60000 to 90000, the Max intensity difference between every glue figure is no more than 15%.Adopt DeCyder6.5 software that the electrophoresis result that scanning obtains is analyzed.
The 2-D DIGE collection of illustrative plates that the freezing sample of salicornia europaeal root system obtains is seen Fig. 2.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.Compare with existing method, in the collection of illustrative plates of the plasmalemma protein solution that the method that adopts invention to provide obtains, albumen is counted more.Choose on glue figure four zones of different molecular weight and different pH scopes and carried out local amplification ratio.Result shows, compare with existing method, in high molecular zone (a, c), lower molecular weight district (b), more protein site (black arrow indication in figure) has all been isolated in pH meta-alkalescence zone (d), and the more plasmalemma protein of cross-film comes across meta-alkalescence district (d) district 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, the 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 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 is consistent with the analytical results of the 2-D DIGE collection of illustrative plates that the freezing sample of salicornia europaeal root system obtains.
The effect of embodiment 6, plasmalemma of plant protein extracting method provided by the invention and existing plasmalemma of plant protein extracting method relatively
1, the plasmalemma protein that the freezing sample of salicornia europaeal root system is obtained proceeds as follows successively:
The plasmalemma protein that the plasmalemma protein of the step 2 extraction of the plasmalemma protein that respectively step 1 of embodiment 3 is extracted, 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 2-D DIGE according to the method for embodiment 5.
2-D DIGE collection of illustrative plates is seen Fig. 3.Find plasmalemma protein that three kinds of methods obtain carry out identical dissolving again after point exist roughly the same distribution, the protein site that the plasmalemma protein that method provided by the invention obtains obtains after dissolving again is (2099) at most, the protein site that the plasmalemma protein that the TCA method obtains obtains after dissolving again is 1652, and the plasmalemma protein that the ammonium acetate method obtains carries out obtaining after dissolving protein site is 1856.
Every glue figure is divided into six districts of a-f by molecular weight and pH, carries out amplification ratio.In a, c, d, e, f five districts, the protein site that the protein site that obtains after 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 the TCA method dissolves again, the plasmalemma protein that obviously obtains more than the ammonium acetate method obtain after dissolving again.
By above-mentioned comparison, adopt method provided by the invention to extract plasmalemma protein many in the more other two kinds of methods of protein site that most of region disconnectings obtain, the plasmalemma protein efficient of extracting is higher, and does not need to regulate protein lysate pH when the DIGE mark, can avoid experimental error.Simultaneously, the albumen species number that this method obtains is more than other two kinds of methods, therefore more is applicable to the 2-D DIGE of plasmalemma protein.
The dissolving again of embodiment 7, plasmalemma protein
1, the 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), supersound process 30s(Ultrasonic Cell Disruptor originates from Shanghai than bright company in ice bath, during work, setting power is 200W, and every work 3s stops 10s; This step must guard against protein solution excess Temperature in ultrasonic procedure and destroys the lysis buffer composition), then be positioned in 4 ℃ of refrigerators, mixed 3h fully dissolves its plasmalemma protein with mixed instrument whirlpool, automatic whirlpool, then room temperature, the centrifugal 30min of 20000g, collect supernatant liquor, be the plasmalemma of plant protein solution.
2, the 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), supersound process 30s(Ultrasonic Cell Disruptor originates from Shanghai than bright company in ice bath, during work, setting power is 200W, and every work 3s stops 10s; This step must guard against protein solution excess Temperature in ultrasonic procedure and destroys the lysis buffer composition), then be positioned in 4 ℃ of refrigerators, in the mixed 3h(practical application in mixed instrument whirlpool, automatic whirlpool, more than 3h all can) its plasmalemma protein is fully dissolved, then room temperature, the centrifugal 30min of 20000g, collect supernatant liquor, be the plasmalemma of plant protein solution.
3, the plasmalemma of plant protein solution that the plasmalemma of plant protein solution that step 1 is obtained and step 2 obtain carries out respectively 2-D DIGE, and method is seen 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) add 400 μ l ice methyl alcohol in the plasmalemma of plant suspension that the step 1 of 100 μ l embodiment 2 obtains, 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 centrifugal 1min of 13000g under room temperature then, this moment, albumen was positioned at the 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 ℃, the centrifugal 5min of 13000g,
Collecting precipitation.
(5) will precipitate with ice methanol wash twice, the plasmalemma of plant albumen that obtains.
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) add 400 μ l ice methyl alcohol in the plasmalemma of plant suspension that the step 1 of 100 μ l embodiment 2 obtains, 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 centrifugal 1min of 13000g under room temperature then, this moment, albumen was positioned at the 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 ℃, the centrifugal 5min of 13000g, collecting precipitation.
(5) will precipitate with ice methanol wash twice, the plasmalemma of plant albumen that obtains.
3, the plasmalemma of plant albumen that the plasmalemma of plant albumen that respectively step 1 is extracted and step 2 are extracted carries out the step 1 of embodiment 4, obtains two kind of plant plasmalemma protein solution.
4, the plasmalemma of plant protein solution is carried out 2-D DIGE according to the method for embodiment 5.
Collection of illustrative plates is all consistent with Fig. 3 A.
Claims (8)
1. method that plasmalemma of plant albumen dissolves again, comprise the steps: plasmalemma of plant albumen is added in lysate, supersound process in ice bath, then the whirlpool is mixed and is made described plasmalemma of plant protein dissolution, last centrifugal collection supernatant liquor is the plasmalemma of plant protein solution; Described lysate adds N-dodecyl-β-D-Maltose glycosides to obtain in the DIGE lysis buffer, and the described N-dodecyl-β-concentration of D-Maltose glycosides in described lysate is 0.5-2g/100mL.
2. the method for claim 1, it is characterized in that: the described N-dodecyl-β-concentration of D-Maltose glycosides in described lysate is 1g/100mL.
3. the method for the preparation of the plasmalemma of plant protein solution that carries out 2-D DIGE, be that plasmalemma of plant albumen is dissolved with the described method of claim 1 or 2 again, obtains the plasmalemma of plant protein solution.
4. method as claimed in claim 3, it is characterized in that: the preparation method of described plasmalemma of plant albumen in turn includes the following steps:
(1) add ice methyl alcohol in plasmalemma of plant suspension, 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) with ice methanol wash precipitation, obtain plasmalemma of plant albumen.
5. as arbitrary described method in claim 1 to 4, it is characterized in that: described plant is monocotyledons or dicotyledons.
6. as arbitrary described method in claim 1 to 4, it is characterized in that: described plant is glycophyte or halophytes.
7. as arbitrary described method in claim 1 to 4, it is characterized in that: described plant is salicornia europaeal or Arabidopis thaliana.
8. the application of arbitrary described method in 2-D DIGE in claim 1 to 7.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105352778A (en) * | 2015-12-18 | 2016-02-24 | 荣成广润水产食品有限公司 | Preparing and processing method for research samples of marine organism proteomics |
| CN105820266A (en) * | 2016-04-20 | 2016-08-03 | 华南农业大学 | Method for efficiently extracting internal mucus layers of arabidopsis thaliana |
| CN106749575A (en) * | 2016-12-27 | 2017-05-31 | 广西民族大学 | A kind of extracting method of the anti-banana anthracnose mycoprotein of Kandelia candel mangrove |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101128586A (en) * | 2004-12-22 | 2008-02-20 | 健泰科生物技术公司 | Methods for producing soluble membrane-spanning proteins |
| CN102183398A (en) * | 2011-02-24 | 2011-09-14 | 中山大学 | Method for external specificity fluorescent staining of methane-oxidizing bacteria |
| WO2012172378A1 (en) * | 2011-06-17 | 2012-12-20 | Isis Innovation Limited | Detection of membrane protein-therapeutic agent complexes by mass spectrometry |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101128586A (en) * | 2004-12-22 | 2008-02-20 | 健泰科生物技术公司 | Methods for producing soluble membrane-spanning proteins |
| CN102183398A (en) * | 2011-02-24 | 2011-09-14 | 中山大学 | Method for external specificity fluorescent staining of methane-oxidizing bacteria |
| WO2012172378A1 (en) * | 2011-06-17 | 2012-12-20 | Isis Innovation Limited | Detection of membrane protein-therapeutic agent complexes by mass spectrometry |
Non-Patent Citations (1)
| Title |
|---|
| 阮松林等: "双向电泳技术研究进展", 《杭州农业科技》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105352778A (en) * | 2015-12-18 | 2016-02-24 | 荣成广润水产食品有限公司 | Preparing and processing method for research samples of marine organism proteomics |
| CN105352778B (en) * | 2015-12-18 | 2018-07-20 | 荣成广润水产食品有限公司 | A kind of preparation processing method for marine protein group study sample |
| CN105820266A (en) * | 2016-04-20 | 2016-08-03 | 华南农业大学 | Method for efficiently extracting internal mucus layers of arabidopsis thaliana |
| CN105820266B (en) * | 2016-04-20 | 2016-12-21 | 华南农业大学 | A kind of method of high efficiency extraction arabidopsis internal layer slime layer |
| CN106749575A (en) * | 2016-12-27 | 2017-05-31 | 广西民族大学 | A kind of extracting method of the anti-banana anthracnose mycoprotein of Kandelia candel mangrove |
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