CN100436476C - Method for preparing dielectrophoresis sample of whole protein of pollen tube in gymnosperm - Google Patents
Method for preparing dielectrophoresis sample of whole protein of pollen tube in gymnosperm Download PDFInfo
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- CN100436476C CN100436476C CNB2006100893098A CN200610089309A CN100436476C CN 100436476 C CN100436476 C CN 100436476C CN B2006100893098 A CNB2006100893098 A CN B2006100893098A CN 200610089309 A CN200610089309 A CN 200610089309A CN 100436476 C CN100436476 C CN 100436476C
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Abstract
The present invention discloses a method for preparing dielectrophoresis samples of pollen tube holoproteins in gymnosperm, which comprises the following steps: 1. pollen tube powder in the gymnosperm is suspended in a trichloroacetic acid solution and is placed at the temperature of 20 DEG C. below zero to 4 DEG C. below zero, and precipitation is collected after the pollen tube powder is centrifugated; the precipitation is suspended in acetone again and is placed at the temperature of 20 DEG C. below zero for 2 to 16 hours, and precipitation is collected after the precipitation is centrifugated; then, 80% of an acetone solution is used for washing, and the crude extractions of the pollen tube holoproteins in gymnosperm are obtained after the acetone is removed; 2. after the crude extractions of the pollen tube holoproteins in gymnosperm, which are obtained by step 1, are dissolved in a schizolysis buffer solution, supernatant fluid is taken by centrifugation to obtain dielectrophoresis sample liquid of pollen tube holoproteins in gymnosperm. The method for isolating and preparing the dielectrophoresis pollen tube holoproteins in gymnosperm has the advantages of quick speed and simple and easy operation; the electrophoretogram of the dielectrophoresis, which has higher quality and preferable repeatability, is obtained; the method for separating and preparing the dielectrophoresis samples of the pollen tube holoproteins in the gymnosperm is convenient to analyze and identify proteins for subsequent biological mass spectrometry and has larger practical application value.
Description
Technical field
The present invention relates to a kind of method for preparing whole protein of pollen tube in gymnosperm, particularly a kind of method for preparing dielectrophoresis sample of whole protein of pollen tube in gymnosperm.
Background technology
Pollen tube has typical polar growth feature as the carrier of male germ unit in the flowering plant fertilization process, thus be that the research cellular form is built up, the idealized model system of the motion of vesica and organoid and signal transduction pathway.Proteomic analysis is the important technical that protein pair cell self changes and environmental stimulus responds in the research pollen tube, helps understanding in depth higher plant syngenesis process and cell polarity growth mechanism.
Selecting suitable sample preparation methods is decision proteome research key of success step.Cell must carry out effective fragmentation, also will remove the degraded that impurity is also avoided protein sample as far as possible simultaneously.Be rich in the impurity that polysaccharide, lipid, nucleic acid etc. disturb the two-dimensional electrophoresis process in the gymnospermae pollen pipe.Also do not prepare effective ways and the technology that the whole protein of pollen tube in gymnosperm sample is used for two-dimensional electrophoresis up to now, thereby cause the relative angiosperm of the proteome research relevant relatively to lag behind with gymnosperm.
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing the dielectrophoresis sample of whole protein of pollen tube in gymnosperm of better quality.
The method for preparing dielectrophoresis sample of whole protein of pollen tube in gymnosperm provided by the present invention may further comprise the steps:
1) the gymnospermae pollen pipe powder is suspended in the 0.1-0.125g/mL trichoroacetic acid(TCA) solution, in-20--4 ℃ following the placement 1-2 hour; Centrifugal back collecting precipitation, this precipitation is resuspended in contains in the acetone soln of mercaptoethanol that volumn concentration is 0.05-0.07%, placed 2-16 hour for-20--4 ℃, centrifugal back collecting precipitation, again with containing the acetone soln washing that mercaptoethanol that volumn concentration is 0.05-0.07% and volumn concentration are 70-80%, obtain the whole protein of pollen tube in gymnosperm crude extract after removing acetone;
2) after the whole protein of pollen tube in gymnosperm crude extract that step 1) is obtained was dissolved in lysis buffer, the centrifuging and taking supernatant obtained dielectrophoresis sample of whole protein of pollen tube in gymnosperm liquid; Described lysis buffer is for containing 5～7mol/L urea (urea), 1-2mol/L thiocarbamide (thiorea), 0.01-0.02g/mL CHAPS (3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonic acid), volumn concentration be 1-2%, pH be 3.5-10 amphotericeledrolyte, the quality percentage composition is the dithiothreitol (DTT) (DTT) of 0.6-1%, the solution of 25-50 μ g/mL phenylmethylsulfonyl fluoride (PMSF).
Described gymnospermae pollen pipe powder can obtain by grinding in liquid nitrogen.
Described 0.1-0.125g/mL trichoroacetic acid(TCA) solution is with containing the acetone soln preparation that volumn concentration is the 0.05-0.07% mercaptoethanol.
The concentration of trichoroacetic acid(TCA) is preferably 0.125g/mL in the described trichoroacetic acid(TCA) solution.
The pollen tube powder suspension was placed 2 hours down in-20 ℃ in trichoroacetic acid(TCA) solution in the described step 1).
Containing volumn concentration in the described step 1) is the resuspended post precipitation of acetone soln of 0.05-0.07% mercaptoethanol, places 2-16 hour in-20 ℃.
In order to obtain better to extract and separating effect, described lysis buffer be preferably contain 7mol/L urea (urea), 2mol/L thiocarbamide (thiorea), 0.02g/ml CHAPS, volumn concentration be 2% amphotericeledrolyte, the quality percentage composition is 1% dithiothreitol (DTT) (DTT), the solution of 50 μ g/mL phenylmethylsulfonyl fluoride (PMSF).
Described step 2) in, the mass volume ratio of whole protein crude extract and lysis buffer can be 120mg/400 μ L-150mg/400 μ L lysis buffer, is preferably 150mg/400 μ L lysis buffer.
In order to make protein dissolution as much as possible in lysis buffer, can lysis buffer 27-37 ℃ of following water-bath 15-30min of whole protein crude extract will be added, at room temperature on the shaking table of 60-150rpm (rotation radius is 25mm), vibrated 1 hour then, 15000-17000g collects supernatant liquor, is dielectrophoresis sample of whole protein of pollen tube in gymnosperm liquid.
The bath temperature of the lysis buffer of described adding whole protein crude extract is preferably 27-29 ℃, and the water-bath time is preferably 30min.
Centrifugal condition in the aforesaid method is the centrifugal 10min at 4 ℃ of 15000rpm (19117g).
Described gymnosperm can be the gymnosperm of Ginkgopsidas such as Song Shangang such as lacebark pine, Korean pine, Chinese pine, dragon spruce, fir, ginkgo and Cycadopsida; Be preferably Song Shangang, especially be preferably the Pinaceae gymnosperm, be preferably white bar or lacebark pine especially.
Method of the present invention adopts trichoroacetic acid(TCA) and acetone effectively to remove the impurity of the interference two-dimensional electrophoresis such as polysaccharide, lipid and nucleic acid in the gymnospermae pollen pipe.Urea in the method lysis buffer of the present invention can make the protein unfolding expose hydrophobic core, can increase the solvability of protein in Stationary pH gradient (IPGs) adhesive tape and be used thiocarbamide, thereby dithiothreitol (DTT) reduction disulfide linkage makes protein reach consoluet state, carrier ampholyte can be removed cyanate ion, be quickened the nucleic acid precipitation when centrifugal, and PMSF then can effectively prevent proteic degraded in the specimen preparation process.
The method for preparing the whole protein of pollen tube in gymnosperm sample of the present invention is quick, simple, and the repeatability that can obtain better quality is the two-dimensional electrophoresis collection of illustrative plates preferably, is convenient to follow-up biological mass spectrometry Analysis and Identification albumen, has bigger actual application value.
Description of drawings
Fig. 1 is white bar pollen tube whole protein sample two-dimensional electrophoresis collection of illustrative plates
Fig. 2 is a lacebark pine pollen tube whole protein sample two-dimensional electrophoresis collection of illustrative plates
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Concentration among the following embodiment if no special instructions, is volume percent (V/V).
Embodiment 1, the white bar pollen tube whole protein dielectrophoresis sample liquid of preparation also carry out Two-dimensional Electrophoresis Analysis
1) filters collection and cultivate the pollen tube that white bar pollen granule obtained in 22 hours, the quick grind into powder of liquid nitrogen with liquid nutrient medium (0.0001g/mL boric acid, 0.0003g/mL nitrocalcite, 0.12g/mL sucrose);
2) powder suspension that step 1) is obtained was placed 2 hours down in-20 ℃ in the acetone soln of the mercaptoethanol that contains 0.125g/ml trichoroacetic acid(TCA) and 0.07%;
3) the centrifugal 10min of 4 ℃ of 15000rpm (19117g), the reject supernatant liquor is resuspended in precipitation in the acetone soln that contains 0.07% mercaptoethanol of-20 ℃ of long-pending precoolings of triploid, places 16 hours for-20 ℃;
4) the centrifugal 10min of 4 ℃ of 15000rpm (19117g), the reject supernatant liquor is resuspended in precipitation in the solution that contains 0.07% mercaptoethanol and 80% acetone, places 1h for-20 ℃;
5) centrifugal reject supernatant liquor obtains precipitation, and repeating step 4 again) 1-2 time.After treating that acetone is evaporated completely, obtain whole protein crude extract dry powder, with the amount dissolving whole protein crude extract dry powder of lysis buffer according to 400 μ L/150mg dry powder, after 29 ℃ of water-bath half an hour, room temperature was vibrated 1 hour on the shaking table of 60-150rpm (rotation radius is 25mm), whole protein crude extract dry powder is fully dissolved, and 17000g room temperature centrifuging and taking supernatant is white bar pollen tube whole protein dielectrophoresis sample liquid afterwards; Lysis buffer is that to contain 7mol/L urea (urea), 2mol/L thiocarbamide (thiorea), 0.02g/ml CHAPS, 2% amphotericeledrolyte (ampholine pH 3.5-10), mass percent be 1% the dithiothreitol (DTT) (DTT) and the solution of 50 μ g/mL phenylmethylsulfonyl fluoride (PMSF).
6) protein that extracts by said process is measured protein concentration with Bradford method ultraviolet spectrophotometer, and the result shows that protein concentration is 4-6 μ g/ μ L in the above-mentioned white bar pollen tube whole protein dielectrophoresis sample liquid that obtains.Carry out two-dimensional electrophoresis as follows: utilize Ettan
TMIPGphor II
TM(General Electric medical treatment group USA) carries out first to isoelectric focusing electrophoresis at 20 ℃ to Isoelectric FocusingSystem.Deposition condition is: 500V electrophoresis 1h, and 1000V electrophoresis 1h subsequently, last 8000V electrophoresis 8.3h, number reaches 64000V * h when always lying prostrate.Stationary pH gradient adhesive tape after will focusing on then 10mL level pad 1 (the 50mmol/LTris-Cl damping fluid, pH 8.8; 6mol/L urea; 30% glycerine, 0.02g/mL SDS, 0.01g/mL dithiothreitol (DTT)) in balance 15min, then 10mL level pad 2 (50mmol/L Tris-Cl damping fluid, pH 8.8; 6mol/L urea; 30% glycerine, 0.02g/mL SDS, 0.25g/mL iodo-acid amide) middle balance 30min.Adhesive tape after the balance is transferred to second on gel.Second utilizes Ettan DALTsixsystem to electrophoresis, and (General Electric medical treatment group USA) carries out (average every clotting glue 25mA constant current electrophoresis, electrophoresis finished when bromine Finland indicator was run out of gel) on the 12.5%SDS-polyacrylamide gel.After gel dyes with Xylene Brilliant Cyanine G R-250, ethanol/acetic acid system decolouring.The result as shown in Figure 1, the result shows that this gymnospermae pollen tubulin sample separation effect is better, and tangible proteolytic degradation does not take place.MW is a protein molecular weight standard among Fig. 1, and unit is kD.
Embodiment 2, separation preparation gymnosperm lacebark pine pollen tube whole protein sample also carry out Two-dimensional Electrophoresis Analysis
1) filters collection and cultivate the pollen tube that the lacebark pine pollen granule obtained in 22 hours, the quick grind into powder of liquid nitrogen with liquid nutrient medium (0.0001g/mL boric acid, 0.0003g/mL nitrocalcite, 0.12g/mL sucrose);
2) powder suspension that step 1) is obtained was placed 1 hour down in-10 ℃ in the acetone soln of the mercaptoethanol that contains 0.1g/ml trichoroacetic acid(TCA) and 0.05%;
3) the centrifugal 10min of 4 ℃ of 15000rpm (19117g), the reject supernatant liquor is resuspended in precipitation in the acetone soln that contains 0.05% mercaptoethanol of-20 ℃ of long-pending precoolings of triploid, places 12 hours for-10 ℃;
4) the centrifugal 10min of 4 ℃ of 15000rpm (19117g), the reject supernatant liquor is resuspended in precipitation in the solution that contains 0.07% mercaptoethanol and 75% acetone, places 1h for-20 ℃;
5) centrifugal reject supernatant liquor obtains precipitation, and repeating step 4 again) 1-2 time.After treating that acetone is evaporated completely, obtain whole protein crude extract dry powder, with the amount dissolving whole protein crude extract dry powder of lysis buffer according to 400 μ L/150mg dry powder, after 29 ℃ of water-bath half an hour, room temperature was vibrated 1 hour on the shaking table of 60-150rpm (rotation radius is 25mm), whole protein crude extract dry powder is fully dissolved, and 17000g room temperature centrifuging and taking supernatant is lacebark pine pollen tube whole protein dielectrophoresis sample liquid afterwards; Lysis buffer is that to contain 5mol/L urea (urea), 1mol/L thiocarbamide (thiorea), 0.01g/ml CHAPS, 1% amphotericeledrolyte (ampholine pH 3.5-10), mass percent be 0.6% the dithiothreitol (DTT) (DTT) and the solution of 25 μ g/mL phenylmethylsulfonyl fluoride (PMSF).
6) protein that extracts by said process is measured protein concentration with Bradford method ultraviolet spectrophotometer, and the result shows that protein concentration is 4-6 μ g/ μ L in the above-mentioned lacebark pine pollen tube whole protein dielectrophoresis sample liquid that obtains.Carry out two-dimensional electrophoresis as follows: utilize Ettan
TMIPGphor II
TM(General Electric medical treatment group USA) carries out first to isoelectric focusing electrophoresis at 20 ℃ to IsoelectricFocusing System.Deposition condition is: 500V electrophoresis 1h, and 1000V electrophoresis 1h subsequently, last 8000V electrophoresis 8.3h, number reaches 64000V * h when always lying prostrate.Stationary pH gradient adhesive tape after will focusing on then 10mL level pad 1 (50mmol/L Tris-Cl damping fluid, pH 8.8; 6mol/L urea; 30% glycerine, 0.02g/mL SDS, 0.01g/mL dithiothreitol (DTT)) in balance 15min, then 10mL level pad 2 (50mmol/L Tris-Cl damping fluid, pH 8.8; 6mol/L urea; 30% glycerine, 0.02g/mL SDS, 0.25g/mL iodo-acid amide) middle balance 30min.Adhesive tape after the balance is transferred to second on gel.Second utilizes Ettan DALTsixsystem to electrophoresis, and (General Electric medical treatment group USA) carries out (average every clotting glue 25mA constant current electrophoresis, electrophoresis finished when bromine Finland indicator was run out of gel) on the 12.5%SDS-polyacrylamide gel.After gel dyes with Xylene Brilliant Cyanine G R-250, ethanol/acetic acid system decolouring.The result as shown in Figure 2, the result shows that the dielectrophoresis sample separating effect of this lacebark pine pollen tube protein sample is better, and tangible proteolytic degradation does not take place.MW is a protein molecular weight standard among Fig. 2, and unit is kD.
Claims (11)
1, a kind of method for preparing dielectrophoresis sample of whole protein of pollen tube in gymnosperm may further comprise the steps:
1) the gymnospermae pollen pipe powder is suspended in the 0.1-0.125g/mL trichoroacetic acid(TCA) solution, in-20--4 ℃ following the placement 1-2 hour; Centrifugal back collecting precipitation, this precipitation is resuspended in contains in the acetone soln of mercaptoethanol that volumn concentration is 0.05-0.07%, placed 2-16 hour for-20--4 ℃, centrifugal back collecting precipitation, again with to contain mercaptoethanol and the volumn concentration that volumn concentration is 0.05-0.07% be the solution washing of the acetone of 70-80%, obtain the whole protein of pollen tube in gymnosperm crude extract after removing acetone;
2) after the whole protein of pollen tube in gymnosperm crude extract that step 1) is obtained was dissolved in lysis buffer, the centrifuging and taking supernatant obtained dielectrophoresis sample of whole protein of pollen tube in gymnosperm liquid; Described lysis buffer is for containing 5-7mol/L urea, the 1-2mol/L thiocarbamide, 0.01-0.02g/mL CHAPS, volumn concentration are 1-2%, pH is the amphotericeledrolyte of 3.5-10, the quality percentage composition is the solution of dithiothreitol (DTT) and the 25-50 μ g/mL phenylmethylsulfonyl fluoride of 0.6-1%.
2, the method for preparing the whole protein of pollen tube in gymnosperm sample according to claim 1 is characterized in that: described 0.1-0.125g/mL trichoroacetic acid(TCA) solution is with containing the acetone soln preparation that volumn concentration is the 0.05-0.07% mercaptoethanol.
3, method according to claim 2 is characterized in that: the concentration of trichoroacetic acid(TCA) is 0.125g/mL in the described trichoroacetic acid(TCA) solution.
4, method according to claim 1 is characterized in that: described lysis buffer be contain 7mol/L urea, 2mol/L thiocarbamide, 0.02g/mL CHAPS, volumn concentration be 2% amphotericeledrolyte, the quality percentage composition is 1% the dithiothreitol (DTT) and the solution of 50 μ g/mL phenylmethylsulfonyl fluoride.
5, method according to claim 1 is characterized in that: the pollen tube powder suspension was placed 2 hours down in-20 ℃ in trichoroacetic acid(TCA) solution in the described step 1); Containing volumn concentration in the described step 1) is the resuspended post precipitation of acetone soln of 0.05-0.07% mercaptoethanol, places 2-16 hour in-20 ℃.
6, method according to claim 1 is characterized in that: described step 2), the mass volume ratio of whole protein crude extract and lysis buffer is a 100mg/400 μ l-150mg/400 μ l lysis buffer.
7, method according to claim 6 is characterized in that: the mass volume ratio of described whole protein crude extract and lysis buffer is a 150mg/400 μ l lysis buffer.
8, method according to claim 1, it is characterized in that: the step that described whole protein crude extract is dissolved in lysis buffer is to add lysis buffer 27-37 ℃ of following water-bath 15-30min of whole protein crude extract, be vibration 1 hour on the shaking table of 25mm, 60-150rpm at room temperature then in rotation radius, 15000-17000g collects supernatant liquor, promptly obtains dielectrophoresis sample of whole protein of pollen tube in gymnosperm liquid.
9, method according to claim 8 is characterized in that: the bath temperature of the lysis buffer of described adding whole protein crude extract is 27-29 ℃, and the water-bath time is 30min.
10, according to arbitrary described method among the claim 1-9, it is characterized in that: described gymnosperm is the gymnosperm of Song Shangang, Ginkgopsida or Cycadopsida; Described loose China fir guiding principle gymnosperm is white bar, blue or green bar, lacebark pine or Chinese pine; Described Ginkgopsida gymnosperm is a ginkgo; Described Cycadopsida gymnosperm is a sago cycas.
11, method according to claim 10 is characterized in that: described gymnosperm is white bar or lacebark pine.
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| CN102241730B (en) * | 2011-05-18 | 2013-06-05 | 淮阴师范学院 | Extraction and dielectrophoresis analytical method for total protein of cattail cauloid |
| CN103265613B (en) * | 2013-03-19 | 2016-05-25 | 中国农业科学院果树研究所 | A kind of method of extracting Malus bark tissue holoprotein |
| CN103900881A (en) * | 2014-04-01 | 2014-07-02 | 淮阴师范学院 | Masson pine needle protein extraction method suitable for two-dimensional electrophoresis |
| CN106810598A (en) * | 2017-03-27 | 2017-06-09 | 许昌学院 | A kind of extracting method of Huaiyin alfalfa |
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Non-Patent Citations (4)
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| Differential display proteomic analysis of Picea meyeri pollengermination and pollen-tube growth after inhibition of actinpolymerization by latrunculin B. Chen Y et al.The Plant Journal,Vol.47 No.2. 2006 |
| Differential display proteomic analysis of Picea meyeri pollengermination and pollen-tube growth after inhibition of actinpolymerization by latrunculin B. Chen Y et al.The Plant Journal,Vol.47 No.2. 2006 * |
| 中国科学院研究生院硕士学位论文"白杄花粉管微丝骨架介导信号传导的蛋白质组研究". 陈艳梅，第32页标题"三、白杄花粉管中微丝解聚后蛋白质组学研究"和3.1.2 实验方法（1）花粉中总蛋白的提取. 2006 |
| 中国科学院研究生院硕士学位论文"白杄花粉管微丝骨架介导信号传导的蛋白质组研究". 陈艳梅，第32页标题"三、白杄花粉管中微丝解聚后蛋白质组学研究"和3.1.2 实验方法(1)花粉中总蛋白的提取. 2006 * |
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