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Plant total protein extracting solution and application thereof

A technology of plant total protein and plant protein, which is applied in the fields of plant peptides, peptide preparation methods, peptide sources, etc., can solve the problems of interference isoelectric focusing, horizontal and vertical stripes, tailing, etc., and achieve the effect of broad application prospects.

Inactive Publication Date: 2010-04-21
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The widespread impurities in plant materials may have little effect on SDS-PAGE electrophoresis, but they will seriously interfere with the isoelectric focusing process, resulting in severe tailing, horizontal and vertical stripes and smearing in 2-DE images

Method used

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  • Plant total protein extracting solution and application thereof
  • Plant total protein extracting solution and application thereof
  • Plant total protein extracting solution and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) 2 g of fresh peanut leaves were ground and crushed with liquid nitrogen, then washed with 10 ml of frozen 10% TCA / acetone (containing 1% (v / v) mercaptoethanol), centrifuged at 10,000 g for 5 min, and the supernatant was discarded. Repeat this process until the supernatant is clear and transparent; add 10ml-20°C pre-cooled acetone to wash twice to remove residual TCA, and then dry it at room temperature in a fume hood;

[0033] (2) Add 3ml of the above protein extract and mix well, let stand at room temperature for 10 minutes to extract protein, centrifuge at 10,000g for 20 minutes, transfer out the supernatant, repeat this process twice;

[0034] (3) Combine all phenol phases, add 5 times the volume of -20°C pre-cooled methanol (containing 0.1M ammonium acetate) and mix evenly, precipitate the protein at -20°C for 10 minutes, wash with methanol three times after centrifugation, and dry at room temperature;

[0035] (4) Dissolve the protein with loading buffer (urea ...

Embodiment 2

[0040] (1) 2 g of fresh young pods of peanuts were ground and pulverized with liquid nitrogen, then washed with 10 ml of frozen TCA / acetone (containing 1% mercaptoethanol), centrifuged at 10,000 g for 5 min, and the supernatant was discarded. Repeat this process until the supernatant is clear and transparent; add 10ml-20°C pre-cooled 80% acetone to wash for more than two times to remove residual TCA, and then dry at room temperature in a fume hood;

[0041] (2) Add 3ml of the above protein extract and mix well, let it stand at room temperature for 10 minutes to extract the protein, centrifuge at 10,000g and transfer the supernatant, repeat this process twice;

[0042] (3) Combine all phenolic phases, add 5 volumes of methanol (containing 0.1M ammonium acetate) to precipitate protein at -20°C for 10 min, centrifuge, wash with methanol three times, and freeze-dry.

[0043] (4) Dissolve protein with loading buffer (urea 7M, thiourea 2M, CHAPS 4%, 0.01% bromophenol blue), quantify...

Embodiment 3

[0055] (1) 2 g of fresh sweet potato leaves were ground and pulverized with liquid nitrogen, washed with 10 ml of frozen TCA / acetone (containing 1% mercaptoethanol), centrifuged at 10,000 g for 5 min, and the supernatant was discarded. Repeat this process until the supernatant is clear and transparent; add 10ml-20°C pre-cooled 80% acetone to wash for more than two times to remove residual TCA, and then dry at room temperature in a fume hood;

[0056] (2) Add 3ml of the above protein extract and mix well, let it stand at room temperature for 10 minutes to extract the protein, centrifuge at 10,000g and transfer the supernatant, repeat this process twice;

[0057] (3) Combine all phenolic phases, add 5 times volume of -20°C pre-cooled methanol (containing 0.1M ammonium acetate) to precipitate protein at -20°C for 10 min, centrifuge and wash with methanol three times, freeze-dry.

[0058] (4) Dissolve the protein with loading buffer (urea 7M, thiourea 2M, CHAPS 4%, 0.01% bromophen...

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Abstract

The invention provides a plant total protein extracting solution and application thereof. The extracting solution is an aqueous solution comprising 80 to 90 percent of redistilled phenol and a reducing agent, wherein the reducing agent is mercaptoethanol or dithiothreitol. Before protein is extracted by using the extracting solution, the impurities such as pigment, grease and the like are removed by trichloroacetic acid / acetone or other methods first according to the properties of samples, and the residual trichloroacetic acid is washed out by using the acetone or other organic solvents; after the samples are dried in a sort, the protein extracting solution is added into the dried samples and is evenly mixed with the samples; and standing the mixture for at least 10 minutes at the room temperature to extract the protein. The method provided by the invention uses phenol to extract the protein directly from dry powder of the plant samples so as to avoid the limit of protein solubility and enable the prepared protein samples to reflect the composition of the protein in a study object comprehensively. The plant total protein extracting solution can be widely used for proteomics study of various plant tissue samples in particular plant leaves, roots, fruits, tubers and the like containing more impurities such as the pigment, amylase and polyphenol.

Description

technical field [0001] The invention relates to a protein extraction method in the field of proteomics research, in particular to a plant total protein extraction liquid and application thereof. Background technique [0002] There are many extraction methods for vegetable protein, the most commonly used are TCA / acetone precipitation and phenol extraction. TCA / acetone can effectively inhibit the activity of various enzymes in the sample, but the obtained protein is often difficult to re-dissolve and contains more impurities, which is not suitable for two-dimensional electrophoresis (2-DE) analysis. The phenol extraction method usually needs to use a buffer solution to dissolve the protein from the plant sample, and then use phenol to extract the protein from the buffer solution. It can be repeatedly extracted with the buffer solution to remove impurities in the phenol phase, and the obtained protein will be relatively Pure, but some protein will be lost due to the limited so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K1/14
Inventor 张二华梁炫强陈小平
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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