CN104478999A - Method of extracting bovine mycoplasma outer membrane protein suitable for proteomics - Google Patents
Method of extracting bovine mycoplasma outer membrane protein suitable for proteomics Download PDFInfo
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- CN104478999A CN104478999A CN201410699960.1A CN201410699960A CN104478999A CN 104478999 A CN104478999 A CN 104478999A CN 201410699960 A CN201410699960 A CN 201410699960A CN 104478999 A CN104478999 A CN 104478999A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
Abstract
The invention relates to a method of extracting a bovine mycoplasma outer membrane protein suitable for proteomics. The method comprises the following steps: culturing and collecting a strain, grinding by liquid nitrogen, carrying out constant-temperature ultrasonic concussion, centrifugally collecting protein precipitate, rinsing with ethanol and acetone, then carrying out centrifugal drying by virtue of vacuum freezing to obtain protein powder, and carrying out cracking treatment; and uniformly mixing the cracked solution, carrying out centrifugation, and maintaining supernatant to obtain the bovine mycoplasma outer membrane protein. According to the method disclosed by the invention, the protein extraction steps and used reagents are optimized, so that the extraction efficiency and purity degree of the protein are greatly improved, and impurities interfering a first dimension and a second dimension in dimensional electrophoresis are effectively removed; compared with the traditional method, the method is simple and convenient, is capable of saving labor and time, is high in protein extraction efficiency and less in disruptors and can be used for obtaining a map with high resolution, good repeatability, uniform protein spot distribution and clear protein spots, and has important significance for research on contagious bovine pleuropneumonia mycoplasma omics and screening research of immune proteins.
Description
Technical field
The present invention relates to a kind of extracting method of outer membrane protein, be specifically related to a kind of Mycoplasma bovis outer membrane protein extracting method being suitable for proteomics, belong to biological technical field.
Background technology
Contagious bovine pleuropneumonia, also known as pleuropneumonia, is a kind of high degree in contact infectious pneumonias of ox caused by thread mycoplasma.This disease with interstitial lung lymphatic vessel, knot hoof tissue and the exudative inflammation of alveolar tissue and serosity cellulosic pleuropneumonia for principal character.Susceptible animal mainly milk cow, beef cattle, ox, buffalo, the yak etc. of this disease.Various ox is different to the susceptibility of this disease, and have difference according to its kind, mode of life and individual resistibility difference, sickness rate is 60 ~ 70%, and case fatality rate is 30 ~ 50%, and other animals and people are without susceptibility.Contagium is sick ox and the ox that carries disease germs mainly, mostly in inapparent infection.There is the trend in rising year by year in China in recent years in this disease, has become one of the most serious disease of harm cattle-raising, caused serious financial loss to China's cattle-raising.
Along with the develop rapidly of modern science and technology, the biological genome order-checking of numerous species completes, and the emphasis of modern life science research transfers to the genome times afterwards comprehensively, i.e. the research of functional genome from Structural genomics research.Protein is the agent of gene function, performs physiological function.In numerous biological mechanism research, many problems can not be completely revealed on gene level, so will study its function, studies from its agent's protein angle.Proteomics is research centre with protein group, and all protein information of systematic study cell or tissue in integral level, has extensive, high-throughout feature.21 century is great development period of life science, and genomics research has entered the post-genomic science epoch, i.e. functional proteomics.The research contents of proteomics widely, mainly comprises the following aspects: the content of several aspects such as structural proteomics, expression proteomics, functional proteomics, Different Proteomics.Two-dimensional gel electrophoresis (2-dementional gel electrophoresis, 2-DE) is protein separation technology ripe, the most classical in current proteomics research.This technology is based upon on the basis of polyacrylamide gel electrophoresis, from these two features of isoelectric point of protein and molecular weight, the protein component of complexity is carried out high-throughout separation, first according to iso-electric point do not coexist Stationary pH gradient immobilized ph gradient strip in isoelectrofocusing (IEF), albumen is carried out first to separation, and then vertical direction is carried out second according to the size of molecular weight and is separated to polyacrylamide gel electrophoresis (SDS-PAGE), realize the separation of horizontal and vertical both direction, improve the sensitivity of albumen sepn.Two-dimensional gel electrophoresis is a kind of protein stripping technique conventional in proteomics research, its step is more, research contents is complicated, mainly comprises: the dialysis desalting, first of the extraction of the collection of raw material and recovery, protein, protein purification, protein is to isoelectrofocusing, second to contents such as polyacrylamide gel electrophoresis, Mass Spectrometric Identification, bioinformatic analysis researchs.
Bidirectional electrophoresis technique is a kind of protein stripping technique conventional in proteomics research, comprises sample preparation, adhesive tape aquation, isoelectrofocusing, polyacrylamide gel electrophoresis, gel fix and the series of steps such as dyeing.At present, 2-DE technology with high-throughput, high resolving power, highly sensitive and the feature such as reproducible, method for protein isolation the most frequently used in proteomics research of still can yet be regarded as.Although 2-DE technology can by most protein separation in tissue or cell or humoral sample, that applies in life science field is also more and more wider, and still there is many problems needs to solve.Isoelectrofocusing is extremely strict to the requirement of sample, if will make isoelectrofocusing procedure ends containing too much impurity in sample.There is a variety of method for extracting protein at present, but do not have a kind of method to be general, therefore will take different measures to different biological tissue.Containing a large amount of interfering substance in bacterium, these material severe jamming isoelectrofocusing and affect the analysis of electrophoretogram.As for extremely acid, pole alkali, excessive too small, the separation of insoluble protein matter and the repeatability of electrophoretogram and resolving power not good, hydrophobic proteins and high molecular weight protein are difficult to proceed to second to electrophoresis, trace modulin is often covered by the structural protein of high-content, these defects are often relevant to the extracting method of albumen, make the application of 2-DE technology in proteomics be restricted to a certain extent.
Summary of the invention
The object of the invention is to: a kind of Mycoplasma bovis outer membrane protein extracting method being suitable for proteomics is provided, the method is easy and simple to handle, protein extraction efficiency is high, impurity is few, do not disturb first to isoelectrofocusing and second to polyacrylamide gel electrophoresis, for the protein science research of the thread mycoplasma of contagious bovine pleuropneumonia, vaccine candidate and protein screening provide technical support and theoretical basis.
Technical scheme of the present invention:
Be suitable for an extracting method for the Mycoplasma bovis outer membrane protein of proteomics, comprise the following steps:
(1) cultivation of thalline and collection, be collected in the bacterium that mycoplasma culture medium grows, through yeast culture, obtain the thalline of freezen protective, for subsequent use;
(2) get freezing thalline, by liquid nitrogen grinding, accurately take a certain amount of thalli powder in centrifuge tube; The protein extract of equivalent is added in centrifuge tube; Fully mixed by gained solution, thermostatic ultrasonic shakes;
(3) at rotating speed be 12000 rpm whizzer in 4 DEG C of centrifugal 15 min, collect supernatant liquor, in supernatant liquor, add the TCA-acetone of 5 times of volumes 10%, be placed in-20 DEG C of precipitates overnight, then be centrifugal 30 min in 12000 rpm whizzers at rotating speed under 4 DEG C of conditions, collect protein precipitation; Containing the Beta-dimercapto base ethanol of 0.5% in described TCA-acetone;
(4) by the described protein precipitation ethanol rinse twice of 95%, then use the Acetone rinse three times of-20 DEG C, with the process of vacuum freezing centrifugal drier, obtain protein powder;
(5) protein powder is added sample dissociation liquid and carry out cracking process;
(6) solution after cracking is fully mixed, in 4 DEG C of centrifugal 15 min in the whizzer of 12000 rpm, retain supernatant liquor, namely obtain Mycoplasma bovis outer membrane protein, this outer membrane protein is stored in the refrigerator of-80 DEG C, for subsequent use.
Described yeast culture process is: get the bacterium that the mycoplasma culture medium of collection grows, with the rotating speed of 2000 rpm centrifugal 10 min in whizzer, remove unnecessary substratum, by the PBS buffer solution for cleaning 3 times of the pH 7.0 of precooling, move in centrifuge tube after cleaning up by bacterial sediment, the amount of bacteria that each centrifuge tube loads is 1/3 of centrifuge tube cumulative volume, unnecessary PBS damping fluid is removed with liquid-transfering gun, use the ParafilmTM mouth of pipe ,-80 DEG C of freezen protective thalline, for subsequent use.
Described a certain amount of thalli powder is 0.1-0.15g; Wherein require during thermostatic ultrasonic concussion: constant temperature 4 DEG C, the concussion time is 10-20 min, and power is 120 W.
Consisting of of protein extract used: the DTT of the urea of 5 M, the thiocarbamide of 4 M, 45 mM, the CHAPS of 6%, the carrier ampholyte solution of 1.2%, the pH value of carrier ampholyte is 3-10, and all the other are pure water.
Consisting of of described sample dissociation liquid: the DTT of the CHAPS of 2%, the urea of 8 M, 50 mM, the tetrabromophenol sulfonphthalein of 0.02%, the carrier ampholyte solution of 2%.
Positive beneficial effect of the present invention:
The extracting method of the present invention to contagious bovine pleuropneumonia mycoplasma outer membrane protein is optimized, and sets up a set of stable, efficient, outer membrane protein extracting method of being suitable for the Mycoplasma bovis of two-dimensional electrophoresis.Compared with traditional method, the method has saving of work and time, easy and simple to handle, and protein extraction efficiency is high, impurity is few, can effectively remove the advantages such as isoelectrofocusing chaff interference; In addition, the method optimizes protein extraction lysate, and liquid nitrogen grinding is combined with ultrasonic vibration process, and intracellular albumen is effectively discharged, and improves extraction efficiency and the quality of albumen.The method is suitable for material that albumen difficulty extracts and the more material of foreign matter content.
The present invention adds the TCA-acetone containing a certain amount of Beta-dimercapto base ethanol in albumen supernatant liquor, can effectively except the impurity such as salt, acidity in Deproteinization; Adopt supercentrifuge centrifugation albumen, make albumen concentrate on sediment fraction, albumen is separated with non-protein and impurity, be beneficial to the further Isolation and purification of albumen; Repeatedly use the ethanol of 95% and the Acetone rinse of-20 DEG C, effectively can remove the impurity in sample, improve the yield of albumen.
Protein extracting method of the present invention, adopts and is added in sample dissociation liquid by protein powder, then except the impurity portion in deproteinize, decontamination effect improving is better; Adopt secondary high speed centrifugation to be separated again, the salt in sample, nucleic acid, pigment, grease impurity can be removed further, make albumen purer; Can ensure that the secondary of albumen dissolves and improves the applied sample amount of sample and carrying out smoothly of isoelectrofocusing.
The protein extracting method of Mycoplasma bovis two-dimensional electrophoresis of the present invention, protein extraction step and agents useful for same are optimized, effectively removes in two-dimensional electrophoresis disturb first to second to impurity, establish a kind of Mycoplasma bovis outer membrane protein extracting method being suitable for proteomics.Method of the present invention eliminates the tedious steps such as the dialysis desalination in traditional method, and whole process is carried out under 4 DEG C of conditions, effectively reduces protein denaturation.
The albumen purity that the present invention extracts is higher, clear, even, reproducible, that resolving power is high electrophoretogram can be obtained, be conducive to the follow-up mass spectroscopy of protein site, for the group research of contagious bovine pleuropneumonia mycoplasma provides technical support, to the screening of Mycoplasma bovis pathogenesis and immune protein and vaccine candidate significant.
Table 1 be the inventive method with other several method in the extraction efficiency of albumen and comparing of purity.
In table, data are this Lung biopsy OD through Different Extraction Method gained under same materials (0.1g)
595nmvalue and final protein concentration.Wherein the unit of protein concentration, OD595 is respectively microgram/microlitre, nanometer, and numerical value is mean value.As can be seen from Table 1, the efficiency that method of the present invention is extracted is higher, and the purity of albumen is higher.
Accompanying drawing explanation
Fig. 1 is the Two-dimensional Gel Electrophoresis of the Mycoplasma bovis outer membrane protein adopting the inventive method to obtain.
Point in Fig. 1 is protein site, and the top of figure is the iso-electric point of albumen, and the right is the molecular mass of albumen, schemes iso-electric point and the molecular weight of known each protein site accordingly.
Embodiment
Further illustrating the present invention with specific examples below, the percentage composition in literary composition, all referring to weight percent as being not particularly illustrated; Agents useful for same is analytical pure, and water is ultrapure water.
Example 1: a kind of Mycoplasma bovis outer membrane protein extracting method being suitable for proteomics, comprises the following steps:
(1) cultivation of thalline and collection, be collected in the bacterium that mycoplasma culture medium grows, centrifugal 10 min of 2000 rpm, remove unnecessary substratum, the PBS(pH 7.0 with precooling) buffer solution for cleaning 3 times; After cleaning up, moved to by bacterial sediment in the centrifuge tube (EP pipe) of 1.5 ml, the amount of bacteria that each EP pipe loads is 1/3 of pipe cumulative volume, about 500 μ l, wash unnecessary PBS damping fluid off with liquid-transfering gun, use the ParafilmTM mouth of pipe, thalline is in-80 DEG C of freezen protective, for subsequent use.
(2) get the freezing thalline of-80 DEG C, liquid nitrogen grinding, accurately take in thalli powder to the 1.5 ml centrifuge tube of a certain amount of (0.1g).
(3) in centrifuge tube, add the protein extract of equivalent (0.1ml), the formula of protein extraction lysate used is: 5 M(mol/l) urea, the thiocarbamide of 4 M, 45 mM(mmol/l) dithiothreitol (DTT) (DTT), 3-[3-(courage amido propyl) dimethylamino] the propanesulfonic acid inner salt (CHAPS) of 6%, the amphotericeledrolyte (pH value is 3-10) of 1.2%.
(4) after above-mentioned solution fully being mixed, constant temperature 4 DEG C of ultrasonic vibration 15 min.
(5) at rotating speed be 12000 rpm whizzer in 4 DEG C of centrifugal 15 min, collect supernatant liquor, 10% TCA-acetone of 5 times of volumes is added in supernatant liquor, wherein contain the Beta-dimercapto base ethanol of 0.5% in TCA-acetone, be placed in-20 DEG C of precipitates overnight, be 4 DEG C of centrifugal 30 min in the whizzer of 12000 rpm at rotating speed, collecting precipitation.
(6) by the protein precipitation that the obtains ethanol rinse twice of 95%, then use the Acetone rinse three times of-20 DEG C, with vacuum freezing centrifugal drier process albumen precipitation, obtain protein powder.
(7) protein powder is added sample dissociation liquid and carries out cracking, consisting of of sample dissociation liquid: the CHAPS of 2%, the urea of 8 M, 50 mM to be DTT, 0.02% be tetrabromophenol sulfonphthalein, 2% carrier ampholyte solution.
(8) fully mixed by above-mentioned solution, in lower 4 DEG C of centrifugal 15 min of 12000 rpm, retain supernatant liquor, remove precipitation, supernatant liquor is stored in the refrigerator of-80 DEG C, for subsequent use.
Example 2: the detection of Mycoplasma bovis outer membrane protein and verification method, detected by the outer membrane protein that example 1 obtains, concrete grammar is as follows:
(1) detection of protein concentration adopts Bradford method, take bovine serum albumin as standard, with aseptic deionized water, bovine serum protein (BSA) is mixed with the standardized solution of different concns, add in Coomassie brilliant G-250 solution, with Coomassie brilliant G-250 staining fluid for contrast, mixing placement 10 min.The light absorption value of solution at 595nm place is measured, drawing standard curve with UV detector.Get testing protein quality sample, OD595 light absorption value under 595nm wavelength, according to the content of protein in typical curve calculation sample.
(2) sample hydration, uses pipettor pipette samples, and along aquation dish, to a left side, the right side linearly adds sample, each 1cm place not application of sample at the two ends of aquation dish, does not have bubble to produce during application of sample.From the refrigerator of-80 DEG C, take out the IPG adhesive tape (18cm pH 3-11) of freezen protective, room temperature places 10min.Distinguish the positive and negative electrode of adhesive tape, peel off the protective membrane of IPG adhesive tape from the side of adhesive tape, glue faces down, and slowly puts into aquation dish, sample solution is not got on the adhesive tape back side, because these solution are not easy to be absorbed by adhesive tape; And movable, avoid producing bubble.If generation bubble, mention one end of adhesive tape gently with tweezers, movable, until bubble gets the gate.In order to prevent liquid evaporation, above adhesive tape, cover the mineral oil of 3ml, cover lid.Aquation under room temperature, places 24h.
(3) first to isoelectrofocusing (IEF), gets two IEF electrode slices, moistening with 7 μ l ultrapure waters.Respectively two electrode slices are placed on the two ends of IPG adhesive tape groove, electrode are pressed in respectively the outer rim of two electrode slices.The adhesive tape of abundant aquation is taken out from aquation dish, use ultrapure water adhesive tape, remove the mineral oil of adhesive tape surface and unabsorbed sample liquid, with tweezers adhesive tape glue faced down and put into adhesive tape groove gently, add 2-3ml mineral oil and cover adhesive tape, focusing dish is moved into and focuses in instrument.Contacted with the anode platform of IPGphor instrument by the tip surface electrode of standard type adhesive tape groove, the flat electrode of adhesive tape groove contacts with the cathode platform of IPGphor instrument.Setting IEF program also starts, and carries out isoelectrofocusing.Isoelectrofocusing program is stopped when voltage and time reach required numerical value.The adhesive tape of isoelectrofocusing carries out balancing immediately, the second to SDS-PAGE electrophoresis, or is positioned in aquation dish by IPG adhesive tape, preserves in-80 DEG C of refrigerators.
(4) balance of IPG adhesive tape, takes out adhesive tape, rinses adhesive tape with aseptic deionized water from focusing dish, removes mineral oil and impurity, is faced up by the glue of adhesive tape and put into balancing drum.By IPG adhesive tape balance twice, each 15 min.In first time level pad, add DTT, make the albumen of sex change be in reduced state, add iodo-acid amide in second time level pad, make the alkylation of protein mercapto.
(component is: the Tris-HCL of 50 mM, 6 M urea, the glycerine of 30% to get 20ml level pad I, the SDS of 2%, the tetrabromophenol sulfonphthalein of 0.002%, the DTT of 0.1g, pH 8.8), each adhesive tape adds 10ml, be placed on horizontal shaker and shake 15min, outwell level pad I, with deionized water rinse IPG adhesive tape 30s, the edge of adhesive tape is placed in 1min on filter paper, to remove unnecessary level pad I.
(component is: the Tris-HCL of 50 mM, the urea of 6 M, the glycerine of 30% to get 20ml level pad II, the SDS of 2%, the tetrabromophenol sulfonphthalein of 0.002%, the iodo-acid amide of 0.25g, pH 8.8), each adhesive tape adds 10ml, be placed on horizontal shaker and shake 15min, outwell level pad II, with deionized water rinse IPG adhesive tape 30s, the edge of adhesive tape is placed in 1min on filter paper, to remove unnecessary level pad II.
(5) second to vertical SDS-PAGE electrophoresis, the acrylamide gel two pieces of preparation 12%.Join the gelating soln of 120ml, every clotting glue uses 60ml.Install encapsulating die, pour gelating soln into, the space of 1cm is stayed on top, binds with Virahol, is exposed to air to reduce gelating soln, keeps glue face smooth.Put into 4 DEG C of room polymerize overnight.Gelating soln proportioning is as follows: 4.9 ml distilled waters, 6 milliliter of 30% acrylamide, 3.8 milliliters of separation gel damping fluids, 150 microlitre 10%SDS, 150 microlitre 10% ammonium persulphates, 6 microlitre Tetramethyl Ethylene Diamines.After layering appears in the liquid of gel and top, show that gel is polymerized completely.After gel solidifies completely, outwell the isopropylcarbinol of gel surface, and with deionized water rinsing glue face.
The transfer of IPG adhesive tape: the IPG adhesive tape after balance is positioned in the gel surface between sheet glass, with a thin chi, IPG adhesive tape is pushed away downwards gently, the bottom of adhesive tape is fully contacted with gel.Note not producing any bubble in the below of adhesive tape.Add molecular weight marker proteins: any one end molecular weight marker proteins adhesive tape being added in gel, makes it contact completely with gel.
Agarose sealing liquid binds: above IPG adhesive tape, adds sealing liquid, makes sealing liquid cover adhesive tape completely, places 5min for 4 DEG C.Sealing liquid is thoroughly solidified.
Electrophoresis: install electrophoretic buffer in electrophoresis chamber, switches on power, and with low voltage (180V) electrophoresis during beginning, walks out completely after IPG adhesive tape until sample, then high voltage (200V).Electrophoresis can be terminated when Bromophenol Blue dye moves to gel bottom margin.After electrophoresis terminates, film is carefully transferred in colouration box fixing, dye after fixing.
As shown in Figure 1, collection of illustrative plates clear background, resolving power are high, protein site is clear, have good repeatability for coloration result, can distinguish that protein site is 987 in figure, meet the needs of subsequent group research.
Be more than preferred embodiment of the present invention, all any equivalent variations of doing according to patent claim of the present invention and modification, all belong to protection scope of the present invention.
Claims (6)
1. be suitable for an extracting method for the Mycoplasma bovis outer membrane protein of proteomics, it is characterized in that, the method comprises the following steps:
(1) cultivation of thalline and collection, be collected in the bacterium that mycoplasma culture medium grows, through yeast culture, obtain the thalline of freezen protective, for subsequent use;
(2) get freezing thalline, by liquid nitrogen grinding, accurately take a certain amount of thalli powder in centrifuge tube; The protein extract of equivalent is added in centrifuge tube; Fully mixed by gained solution, thermostatic ultrasonic shakes;
(3) at rotating speed be 12000 rpm whizzer in 4 DEG C of centrifugal 15 min, collect supernatant liquor, in supernatant liquor, add the TCA-acetone of 5 times of volumes 10%, be placed in-20 DEG C of precipitates overnight, then be centrifugal 30 min in 12000 rpm whizzers at rotating speed under 4 DEG C of conditions, collect protein precipitation; Containing the Beta-dimercapto base ethanol of 0.5% in described TCA-acetone;
(4) by the described protein precipitation ethanol rinse twice of 95%, then use the Acetone rinse three times of-20 DEG C, with the process of vacuum freezing centrifugal drier, obtain protein powder;
(5) protein powder is added sample dissociation liquid and carry out cracking process;
(6) solution after cracking is fully mixed, in 4 DEG C of centrifugal 15 min in the whizzer of 12000 rpm, retain supernatant liquor, namely obtain Mycoplasma bovis outer membrane protein, this outer membrane protein is stored in the refrigerator of-80 DEG C, for subsequent use.
2. extracting method according to claim 1, it is characterized in that, described yeast culture process is: get the bacterium that the mycoplasma culture medium of collection grows, with the rotating speed of 2000 rpm centrifugal 10 min in whizzer, remove unnecessary substratum, by the PBS buffer solution for cleaning 3 times of the pH 7.0 of precooling, after cleaning up, bacterial sediment is moved in centrifuge tube, the amount of bacteria that each centrifuge tube loads is 1/3 of centrifuge tube cumulative volume, unnecessary PBS damping fluid is removed with liquid-transfering gun, use the ParafilmTM mouth of pipe ,-80 DEG C of freezen protective thalline, for subsequent use.
3. extracting method according to claim 1, is characterized in that, described a certain amount of thalli powder is 0.1-0.15g.
4. extracting method according to claim 1, is characterized in that, wherein requires during thermostatic ultrasonic concussion: constant temperature 4 DEG C, the concussion time is 10-20 min, and power is 120 W.
5. extracting method according to claim 1, it is characterized in that, consisting of of protein extract used: the DTT of the urea of 5 M, the thiocarbamide of 4 M, 45 mM, the CHAPS of 6%, the carrier ampholyte solution of 1.2%, the pH value of carrier ampholyte is 3-10, and all the other are pure water.
6. extracting method according to claim 1, is characterized in that, consisting of of described sample dissociation liquid: the DTT of the CHAPS of 2%, the urea of 8 M, 50 mM, the tetrabromophenol sulfonphthalein of 0.02%, the carrier ampholyte solution of 2%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105486561A (en) * | 2016-01-14 | 2016-04-13 | 青海省农林科学院 | Broad bean leaf proteomics analysis sample preparation method |
| CN106167511A (en) * | 2016-08-04 | 2016-11-30 | 中国农业科学院兰州兽医研究所 | A kind of extracting method of Mycoplasma bovis biofilm total protein |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280002A (en) * | 2008-05-28 | 2008-10-08 | 河北农业大学 | Extracting method of jujube protein suitable for dielectrophoresis |
-
2014
- 2014-11-28 CN CN201410699960.1A patent/CN104478999A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280002A (en) * | 2008-05-28 | 2008-10-08 | 河北农业大学 | Extracting method of jujube protein suitable for dielectrophoresis |
Non-Patent Citations (5)
| Title |
|---|
| 刘维全: "《动物生物化学实验指导 第3版》", 31 March 2008 * |
| 夏其昌等: "《蛋白质化学与蛋白质组学》", 30 April 2004 * |
| 崔永锋等: "三氯醋酸-丙酮沉淀法用于骨组织蛋白的提取", 《中国医学科学院学报》 * |
| 陈捷: "《农业生物蛋白质组学》", 31 May 2009 * |
| 陈维等: "牛支原体蛋白质组学双向电泳技术的建立及初步分析", 《东北农业大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105486561A (en) * | 2016-01-14 | 2016-04-13 | 青海省农林科学院 | Broad bean leaf proteomics analysis sample preparation method |
| CN106167511A (en) * | 2016-08-04 | 2016-11-30 | 中国农业科学院兰州兽医研究所 | A kind of extracting method of Mycoplasma bovis biofilm total protein |
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Application publication date: 20150401 |