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Method for preparing aphid protein sample applicable to dielectrophoresis

A two-dimensional electrophoresis and sample preparation technology, which is applied in the field of farmland pest control, can solve the problems of proteome research lag, complex components, and low protein content of aphids, and achieve high practical value, good repeatability, and fast sample results.

Inactive Publication Date: 2012-10-10
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein content of aphids is relatively low, the composition is complex, and it is rich in interfering substances such as lipids, nucleic acids, and polysaccharides. These substances bring difficulties to protein quantification and further analysis
In addition, some skeletal proteins, actin, etc. are not easy to dissolve, which makes the protein extraction of aphids very difficult. Therefore, it is necessary to establish an extraction method for aphid proteins suitable for electrophoresis analysis.
So far, there is no effective method and technology for preparing aphid protein samples for two-dimensional electrophoresis, which leads to a lag in proteome research related to aphids compared to other insects

Method used

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  • Method for preparing aphid protein sample applicable to dielectrophoresis
  • Method for preparing aphid protein sample applicable to dielectrophoresis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Preparation of a two-dimensional electrophoresis sample of the aphid protein and performing two-dimensional electrophoresis analysis

[0021] 1. Collect 3-day-old Aphid aphid adults, add liquid nitrogen and quickly grind them into powder; the weight ratio of aphids to liquid nitrogen is 1:100;

[0022] 2. Put the powder obtained in step 1 into a microcentrifuge tube, add 1.5ml of cold acetone for every 10mg of powder, mix and shake for 30s, and centrifuge at 10,000×g for 3min at 4°C to obtain a precipitate;

[0023] 3. Wash the precipitate obtained in step 2 once with cold acetone, then transfer it to a mortar and dry it at room temperature for 20 minutes, add quartz sand to the dried precipitate at a weight ratio of 4:1, and then add liquid nitrogen at a weight ratio of 1:100 to grind again , transfer the powder to a new centrifuge tube;

[0024] 4. Add an equal volume of saturated phenol solution and SDS buffer to the powder obtained in step 3, and the...

Embodiment 2

[0032] Example 2, separation and preparation of soybean aphid protein samples and two-dimensional electrophoresis analysis

[0033] 1. Collect 3-day-old soybean aphid adults, and quickly grind them into powder after adding liquid nitrogen; the weight ratio of aphids to liquid nitrogen is 1:100;

[0034] 2. Put the powder obtained in step 1 into a microcentrifuge tube, add 1.5ml of cold acetone for every 10mg of powder, mix and shake for 30s, and centrifuge at 10,000×g for 3min at 4°C to obtain a precipitate;

[0035] 3. Wash the precipitate obtained in step 2 once with cold acetone, transfer the precipitate to a mortar and dry at room temperature for 20 minutes, add quartz sand to the dried precipitate at a weight ratio of 4:1, and then add liquid nitrogen at a weight ratio of 1:100 again Grind, then transfer the powder to a new centrifuge tube;

[0036] 4. Add an equal volume of saturated phenol solution and SDS buffer to the powder obtained in step 3, the SDS buffer is 30...

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Abstract

The invention relates to a method for preparing an aphid protein sample applicable to dielectrophoresis, belonging to the field of agricultural pest prevention and control technologies. The method comprises the following steps of: adding aphids into liquid nitrogen, grinding the aphids and the liquid nitrogen together into powder, adding 1.5 ml of cold acetone, evenly mixing, shaking, and centrifuging to collect a precipitate; putting the dried precipitate into liquid nitrogen and quartz sand, then grinding again, and then, adding a saturated phenol solution and an SDS (Sodium Dodecyl Sulfate) buffer solution of the same volume; adding a methanol-dissolved 0.1M ammonium acetate solution of a 5 times volume that of a phenol phase sucked after centrifuging into the phenol phase, putting the mixture into a refrigerator of -20 DEG C for precipitating for 2 hours, washing the precipitate by using the methanol-dissolved 0.1M ammonium acetate solution firstly, and then washing the precipitate by using 80% cold acetone so as to obtain an aphid protein crude extract; and dissolving the aphid protein crude extract into a hydration / pyrolysis buffer solution, then centrifuging, and collecting an supernatant, thereby obtaining an aphid protein dielectrophoresis sample. The method has the advantages that the preparation of the aphid protein sample is simple and fast, the dielectrophoretogram with high quality and good repeatability can be obtained, and the analysis and identification of proteins through a biological mass spectrometry are facilitated. The method is widely applied to aphid proteomics research.

Description

technical field [0001] The invention belongs to the technical field of farmland pest prevention and control, and in particular relates to a method for preparing an aphid protein sample suitable for two-dimensional electrophoresis. Background technique [0002] Aphids, also known as greasy insects, are one of the important pests of crops, which seriously affect the yield and quality of crops, not only cause direct economic losses, but also spread various viral diseases, and are one of the frequent pests. The starting point of aphid development is low, and the duration of generation development is short. In particular, climate warming has a significant impact on the dynamics of aphid populations. An increase in the annual average temperature of 2°C will increase the number of aphids with a very short reproductive cycle by 4-5 generations a year. With global warming, aphids can tolerate higher temperatures, their fecundity and adaptability are significantly enhanced, and the da...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
Inventor 席景会潘怡欧王军孙宇王滔张桦杨巽王玉
Owner JILIN UNIV
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