CN102928278A - Asiatic corn borer larva plasma protein sample preparation method suitable for dimensional electrophoresis - Google Patents
Asiatic corn borer larva plasma protein sample preparation method suitable for dimensional electrophoresis Download PDFInfo
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- CN102928278A CN102928278A CN201210450768XA CN201210450768A CN102928278A CN 102928278 A CN102928278 A CN 102928278A CN 201210450768X A CN201210450768X A CN 201210450768XA CN 201210450768 A CN201210450768 A CN 201210450768A CN 102928278 A CN102928278 A CN 102928278A
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Abstract
An Asiatic corn borer larva plasma protein sample preparation method suitable for dimensional electrophoresis belongs to the technical field of preventing and controlling injurious insects in farmlands. The Asiatic corn borer larva plasma protein sample preparation method comprises the steps that the body surface of Asiatic corn borer larva is sterilized to collect hemolymph, 10mg fo N-phenylthiourea is added into per 200ul of hemolymph, the hemolymph is centrifuged to take supernatant to be separated and packed, 80ul of 50% polyethylene glycol (PEG) is added into each tube, the final concentration is 40%, the hemolymph is shocked and evenly mixed to be in ice bath for 1 hour, the hemolymph is centrifuged to remove supernatant, four times of volume of cold acetone suspension is added into sediment to be shocked and evenly mixed to be in ice bath for 30 minutes, and crude extract of Asiatic core borer larva plasma proteins are dissolved into hydration/crack buffer fluid to be centrifuged to take supernatant to obtain Asiatic corn borer larva plasma protein dimensional electrophoresis sample fluid. The preparation method can prepare Asiatic core borer larva plasma protein samples quickly and easily, can obtain high-quality dimensional electrophoresis patterns with good repeatability and facilitates follow-up researches on biomass spectrometry analysis and identification proteins. The preparation method can be widely applied to researches on the Asiatic core bore larva plasma proteins and has high practical value.
Description
Technical field
The invention belongs to agricultural pests prevention and control technical field, be specifically related to a kind of Ostrinia furnacalis larvae plasma proteins sample preparation methods that is applicable to dielectrophoresis.
Background technology
Ostrinia furnacalis claims again borer, belongs to the Lepidoptera Pyralidae, is the Major Pests of corn, the crops such as Chinese sorghum and millet of also causing harm.Corn borer can endanger milpa each position on the ground, makes the part loss of function of being injured, and reduces grain yield.North Spring Maize Area is caused harm because of corn borer, the general time underproduction about 10%, the outbreak year underproduction 20%～30%.Only cause harm because of corn borer be lost in more than 500,000,000 kilograms every year in Jilin Province.
Enforcement and propelling along with the Human Genome Project, life science has entered the genome times afterwards comprehensively, the research of proteomics is the feature that life science enters rear era gene, to comprehensive and deep understanding be arranged to the complicated activity of life, must be whole, dynamically, protein is studied on the level of network, can say that carrying out of proteome research be not only the milestone that life science enters the genome times afterwards comprehensively, also be one of core content of genome times afterwards comprehensively life science.As core, sample preparation and dissolving are concerning the effect of 2-DE with bidirectional electrophoresis technique in the development of proteome research, and target is to improve as far as possible resolution.
Because insect hemolymph is the potpourri of blood and lymph liquid, in the open cycle system of insect, circulate, being the important place of insect metabolism, also is the place of various material storage and exchange in the metabolic process, the defence of insect, freeze proof, immune, damage is replied etc., and the aspect plays an important role.The insect protein preparation method is more, but yet there are no report for the preparation method of Ostrinia furnacalis larvae plasma sample (particularly being suitable for the Ostrinia furnacalis larvae plasma proteins sample of Two-dimensional Electrophoresis Analysis).
Summary of the invention
The purpose of this invention is to provide a kind of Ostrinia furnacalis larvae plasma proteins sample preparation methods with better quality that is applicable to dielectrophoresis.
The present invention includes the following step:
1. with 75% alcohol Ostrinia furnacalis larvae is carried out the body surface sterilization, at-20 ℃ of freezing 10min;
2. taking-up Ostrinia furnacalis larvae, do not destroy under the gastral prerequisite not pushing belly, with the aseptic larva abdominal foot of cutting, draw transparent hemolymph with the liquid-transfering gun of 10ul again, put it into fast in the centrifuge tube on ice, it is oxidized for preventing hemolymph putting into fast;
3. the Ostrinia furnacalis after will drawing is put into the taper centrifuge tube that foraminate 0.5ml is established in the bottom, again the taper centrifuge tube is inserted in the 1.5ml centrifuge tube, behind the centrifugal 10min of 8000 * g under 4 ℃ of conditions, collect hemolymph from 1.5ml centrifuge tube bottom, add the 10mgN-phenylthiourea in every 200ul hemolymph;
4. will collect good hemolymph centrifugal 5min of 5000 * g under 4 ℃ of conditions, and get supernatant, and by every pipe 20ul packing, add the PEG of 80ul 50% in every pipe, the final concentration that makes PEG is 40%, and concussion mixing 30s is put ice bath 1h on ice;
5. the centrifugal 15min of 15000 * g under 4 ℃ of conditions abandons supernatant, and the cold acetone that adds 600ul100% in every pipe precipitation suspends and shakes mixing, puts ice bath 30min on ice;
6. the centrifugal 15min of 15000 * g under 4 ℃ of conditions, abandon supernatant, add 600ul 80% cold acetone suspension concussion mixing in every pipe precipitation, put ice bath 30min on ice, the centrifugal 15min of 15000 * g under 4 ℃ of conditions, the dry 1min of traditional vacuum obtains the crude extract of Ostrinia furnacalis larvae plasma proteins;
7. Ostrinia furnacalis larvae plasma proteins crude extract is mixed with aquation/lysis buffer, bathe 30min at 30 ℃ of Water Unders, promote the dissolving of albumen, then the centrifugal 5min of 15000 * g under 4 ℃ of conditions, collect supernatant, namely obtain being applicable to the Ostrinia furnacalis larvae plasma proteins sample liquid of dielectrophoresis.
The mass volume ratio of the Ostrinia furnacalis larvae plasma proteins crude extract described in the step 6 and 7 and aquation/lysis buffer is 100mg/100uL-300mg/100uL.
Aquation/lysis buffer described in the step 7 is the solution that contains 5mol/L urea, 2mol/L thiocarbamide, 2%CHAPS, 20mM dithiothreitol (DTT), 0.002% bromophenol blue and 2%IPG damping fluid.
The present invention adopts the PEG method to extract albumen, has avoided the impact of the interfering materials such as blood sugar, blood fat, organic acid, pigment on dielectrophoresis.Urea in method aquation/lysis buffer of the present invention can make the protein unfolding expose hydrophobic core, can increase the dissolubility of protein in Stationary pH gradient (IPG) adhesive tape and be used thiocarbamide; The dithiothreitol (DTT) Reduction of Disulfide, thus make protein reach consoluet state; The IPG damping fluid can be removed cyanate ion, accelerate the nucleic acid precipitation when centrifugal.
The preparation method of the Ostrinia furnacalis larvae plasma proteins sample that is applicable to dielectrophoresis of the present invention is quick, simple, can obtain the Two-dimensional Gel Electrophoresis of high-quality, good reproducibility, be convenient to carry out the research that generate subsequent material analysis of spectrum is identified albumen, the present invention can be widely used in the research of Ostrinia furnacalis larvae Plasma Proteomics, has higher practical value.
Description of drawings
Fig. 1 is the Two-dimensional Gel Electrophoresis of Ostrinia furnacalis larvae plasma proteins sample
Wherein: MW is protein molecular weight standard, and unit is kDa.
Embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method; Concentration among the embodiment if no special instructions, is percent by volume (V/V).
Embodiment prepares Ostrinia furnacalis larvae plasma proteins dielectrophoresis sample and carries out Two-dimensional Electrophoresis Analysis
1) collect 5 age Ostrinia furnacalis larvae, with 75% alcohol Ostrinia furnacalis larvae is carried out the body surface sterilization, then be placed on the aseptic filter paper sheet and blot alcohol, with the larva after sterilizing at-20 ℃ of frost anesthesia 10min;
2) larva is taken out, do not destroying under the gastral prerequisite, with the aseptic larva abdominal foot of cutting, the extruding belly is drawn transparent hemolymph with the liquid-transfering gun of 10ul again, and (it is fast that attention speed is wanted to put into centrifuge tube, otherwise the easy oxidation of hemolymph, and carry out on ice);
3) Ostrinia furnacalis after will drawing is put into the taper centrifuge tube that the bottom is provided with the 0.5ml of 5 apertures, again the taper centrifuge tube is inserted in the 1.5ml centrifuge tube, behind the centrifugal 10min of 8000 * g under 4 ℃ of conditions, collect hemolymph from 1.5ml centrifuge tube bottom, add the 10mgN-phenylthiourea in every 200ul hemolymph;
4) will collect good hemolymph centrifugal 5min of 5000 * g under 4 ℃ of conditions, and get supernatant, and by every pipe 20ul packing, add the PEG (polyglycol) of 80ul 50% in every pipe, the final concentration that makes PEG is 40%, and concussion mixing 30s is placed on ice bath 1h on ice;
5) the centrifugal 15min of 15000 * g under 4 ℃ of conditions abandons supernatant, and the cold acetone that adds 600ul100% in every pipe precipitation suspends and shakes mixing, is placed on ice bath 30min on ice;
6) the centrifugal 15min of 15000 * g under 4 ℃ of conditions, abandon supernatant, add 600ul 80% cold acetone suspension concussion mixing in every pipe precipitation, be placed on ice bath 30min on ice, the centrifugal 15min of 15000 * g under 4 ℃ of conditions, the dry 1min of traditional vacuum obtains the crude extract of Ostrinia furnacalis larvae plasma proteins;
7) Ostrinia furnacalis larvae plasma proteins crude extract is mixed with aquation/lysis buffer, bathe 30min at 30 ℃ of Water Unders, then the centrifugal 5min of 15000 * g under 4 ℃ of conditions, supernatant is got in collection, namely obtain being applicable to the Ostrinia furnacalis larvae plasma proteins sample liquid of dielectrophoresis, the mass volume ratio of described Ostrinia furnacalis larvae plasma proteins crude extract and aquation/lysis buffer is 100mg/100uL-300mg/100uL.
Described aquation/lysis buffer is for containing 5mol/L urea, 2mol/L thiocarbamide, 2%CHAPS (3-[3-Cholamidopropyl) dimethylammonio] propanesulfonic acid), the solution of 20mM dithiothreitol (DTT) (DTT), 0.002% bromophenol blue and 2%IPG damping fluid;
8) solution that adds the aquation/lysis buffer of protein crude extract administration is put into water-bath, and bath temperature is 30 ℃, and the time is 30min, and 15000 * g centrifuging and taking supernatant under 4 ℃ of conditions is the Ostrinia furnacalis larvae plasma proteins sample for dielectrophoresis;
9) albumen that extracts by said process is measured protein concentration with Bradford method ultraviolet spectrophotometer, and the result shows that protein concentration is 2-4ug/uL in the dielectrophoresis Ostrinia furnacalis larvae plasma proteins sample obtained above.
Carry out as follows dielectrophoresis: (General Electric's Medical Group USA) carries out first to isoelectric focusing electrophoresis (IEF) at 20 ℃ to utilize Ettan IPGphor III soelectric Focusing System.Deposition condition is: 500V electrophoresis 1h, and 1000V electrophoresis 1h subsequently, 4000V electrophoresis 1h, number was 40000Vh when last 8000V reached total volt.Then the Stationary pH gradient adhesive tape (IPG adhesive tape) after will focusing on is at 10mL level pad 1 (50mmol/L Tris-Cl damping fluid, pH8.8; 6mol/Lurea; 30% glycerine, 0.02g/mL SDS, 0.01g/mL dithiothreitol (DTT)) middle balance 15min, then at 10ml level pad 2 (50mmol/L Tris-Cl damping fluid, pH8.8; 6mol/L urea; 30% glycerine, 0.02g/mL SDS, 0.25g/mL iodoacetamide) middle balance 15min.Adhesive tape after the balance is transferred to second on gel, second utilizes EttanDALTsix system (General Electric's Medical Group to electrophoresis, USA) carry out (average every clotting glue 30mA constant current electrophoresis, electrophoresis finished when bromophenol blue indicator was run out of gel) at the 12.5%SDS-polyacrylamide gel.Gel adopts silver nitrate method staining, the result as shown in Figure 1, the result shows that Ostrinia furnacalis larvae plasma proteins sample separation effect is better, and obvious protein degradation does not occur.
Claims (3)
1. an Ostrinia furnacalis larvae plasma proteins sample preparation methods that is applicable to dielectrophoresis is characterized in that comprising the following steps:
1) with 75% alcohol Ostrinia furnacalis larvae is carried out the body surface sterilization, at-20 ℃ of freezing 10min;
2) take out Ostrinia furnacalis larvae, do not destroying under the gastral prerequisite, with the aseptic larva abdominal foot of cutting, the extruding belly is drawn transparent hemolymph with the liquid-transfering gun of 10ul again, puts it into fast in the centrifuge tube on ice;
3) Ostrinia furnacalis after will drawing is put into the taper centrifuge tube that foraminate 0.5ml is established in the bottom, again the taper centrifuge tube is inserted in the 1.5ml centrifuge tube, behind the centrifugal 10min of 8000 * g under 4 ℃ of conditions, collect hemolymph from 1.5ml centrifuge tube bottom, add the 10mgN-phenylthiourea in every 200ul hemolymph;
4) will collect good hemolymph centrifugal 5min of 5000 * g under 4 ℃ of conditions, and get supernatant, and by every pipe 20ul packing, add the PEG of 80ul 50% in every pipe, the final concentration that makes PEG is 40%, and concussion mixing 30s is put ice bath 1h on ice;
5) the centrifugal 15min of 15000 * g under 4 ℃ of conditions abandons supernatant, and the cold acetone that adds 600ul100% in every pipe precipitation suspends and shakes mixing, puts ice bath 30min on ice;
6) the centrifugal 15min of 15000 * g under 4 ℃ of conditions, abandon supernatant, add 600ul 80% cold acetone suspension concussion mixing in every pipe precipitation, put ice bath 30min on ice, the centrifugal 15min of 15000 * g under 4 ℃ of conditions, the dry 1min of traditional vacuum obtains the crude extract of Ostrinia furnacalis larvae plasma proteins;
7) Ostrinia furnacalis larvae plasma proteins crude extract is mixed with aquation/lysis buffer, bathe 30min at 30 ℃ of Water Unders, promote the dissolving of albumen, then the centrifugal 5min of 15000 * g under 4 ℃ of conditions, collect supernatant, namely obtain being applicable to the Ostrinia furnacalis larvae plasma proteins sample liquid of dielectrophoresis.
2. by the Ostrinia furnacalis larvae plasma proteins sample preparation methods that is applicable to dielectrophoresis claimed in claim 1, the mass volume ratio that it is characterized in that described Ostrinia furnacalis larvae plasma proteins crude extract and aquation/lysis buffer is 100mg/100uL-300mg/100uL.
3. by the Ostrinia furnacalis larvae plasma proteins sample preparation methods that is applicable to dielectrophoresis claimed in claim 1, it is characterized in that described aquation/lysis buffer is the solution that contains 5mol/L urea, 2mol/L thiocarbamide, 2%CHAPS, 20mM dithiothreitol (DTT), 0.002% bromophenol blue and 2%IPG damping fluid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727436A (en) * | 2017-10-18 | 2018-02-23 | 太原师范学院 | A kind of small insects hemolymph collection procedure |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020137710A1 (en) * | 1999-11-05 | 2002-09-26 | Leo Liu | Insect control agent |
| CN102382166A (en) * | 2011-11-22 | 2012-03-21 | 福建农林大学 | Two-dimensional electrophoresis method for separating protein from kenaf leaf efficiently and stably |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020137710A1 (en) * | 1999-11-05 | 2002-09-26 | Leo Liu | Insect control agent |
| CN102382166A (en) * | 2011-11-22 | 2012-03-21 | 福建农林大学 | Two-dimensional electrophoresis method for separating protein from kenaf leaf efficiently and stably |
Non-Patent Citations (3)
| Title |
|---|
| THI THUY AN NGUYEN: "《Proteomic profiling of aphid Macrosiphum euphorbiae responses to host-plant-mediated stress induced by defoliation and water deficit》", 《JOURNAL OF INSECT PHYSIOLOGY》 * |
| 安少利等: "《蚜虫全蛋白提取方法的比较研究》", 《环境昆虫学报》 * |
| 曾璐: "《稻纵卷叶螟体内储存蛋白的研究以及常用药剂防效评价》", 《万方学位论文全文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727436A (en) * | 2017-10-18 | 2018-02-23 | 太原师范学院 | A kind of small insects hemolymph collection procedure |
| CN107727436B (en) * | 2017-10-18 | 2020-08-21 | 太原师范学院 | Small insect hemolymph collection method |
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Application publication date: 20130213 |