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Extraction method of soybean seed total protein and its special reagent

A soybean seed and protein technology, applied in the fields of peptide preparation, chemical instruments and methods, organic chemistry, etc., can solve the problem of hindering the extraction of high-quality protein samples, affecting the number of high-definition protein spots, and affecting the separation effect of 2-DE gel and other problems, to achieve the effect of high purity and great application value

Inactive Publication Date: 2016-08-17
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The widespread presence of interfering substances such as soybean secondary metabolites, lipids, a large amount of carbohydrates, and phenolic mixtures not only hinders the extraction of high-quality protein samples, but also affects the separation effect of 2-DE gels, leading to streaks and stains, affecting high The main reason for the number of clear protein spots

Method used

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  • Extraction method of soybean seed total protein and its special reagent
  • Extraction method of soybean seed total protein and its special reagent
  • Extraction method of soybean seed total protein and its special reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the extraction of soybean seed total protein

[0033] One, extraction method and reagent of the present invention

[0034] Take 0.4g of mature soybean seeds Zhonghuang 13 (Wang Lianzheng, Zhao Rongjuan, Agricultural Science and Technology Communications, 2005, 6:40), crush it, add 1.2ml of solution S1, grind on ice for 10 minutes, then add 1.2ml of solution S2, continue grinding for 10 minutes Minutes; transfer all the grinds to a centrifuge tube, centrifuge at 10,000g at 4°C for 10 minutes; add trichloroacetic acid with a final concentration of 10% to the supernatant, and place on ice for half an hour; centrifuge at 15,000g at 4°C for 10 minutes , take the precipitate and add 2mL solution S3 for washing, wash four times in total (multiple times of large-volume washing of the precipitate can remove impurities in the protein to the greatest extent), dry the precipitate in an ultra-clean bench to obtain total protein Ⅰ, -20°C save.

[0035] The solvent of ...

Embodiment 2

[0042] Example 2, protein two-dimensional electrophoresis, staining and gel scanning analysis

[0043] The total protein I, II and III obtained in Example 1 were subjected to protein two-dimensional electrophoresis, staining and gel scanning analysis respectively, and the results were as follows: figure 1 , figure 2 and image 3 shown. figure 1 The protein spots in the middle are well dispersed, with fewer horizontal and vertical lines, and more protein spots. A total of 900 protein spots were obtained; due to the generation of more air bubbles during grinding, the protein was oxidized and degraded. figure 2 In the results of two-dimensional electrophoresis, there were many horizontal and vertical stripes, and a total of 530 protein spots were obtained; image 3 The CCP obtained 410 protein spots.

[0044] Two-dimensional electrophoresis and gel staining of the above protein obtained Figure 1-3 The specific method of the result is as follows:

[0045] 1. First dimensi...

Embodiment 3

[0059] Embodiment 3, the mass spectrometry analysis of protein spot

[0060] The total protein I obtained in Example 1 was subjected to electrophoresis and analysis in Example 2. The protein spots (a total of 900) were analyzed by mass spectrometry, and a total of 363 protein spots were identified, of which 126 protein spots were identified using MALDI TOF MS. Using MALDI-TOF / TOF to identify 172 protein spots, using LC-MS / MS to identify 65 protein spots, the specific method is as follows:

[0061] 1. In-gel enzymatic hydrolysis

[0062] Cut the gel block containing the target protein spot into 1mm 2 Left and right pieces, add decolorization solution (50% acetonitrile, 50mM NH 4 HCO 3 ) 800 μl, decolorize at 40°C for 2 hours, and replace the decolorization solution once in the middle. Absorb the decolorization solution, rinse with 200 μl HPLC water, shake for 1 min, then absorb the HPLC water, then rinse with 200 μl HPLC water, shake for 1 min. Blot off the HPLC water, dry...

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Abstract

The invention discloses a method for extracting total protein from soybean seeds and dedicated reagents for the method. The method comprises the following steps: (1) mixing soybean seeds and a solution S1 according to a ratio of 1 g: 3 mL, extracting the protein, collecting all the substances so as to obtain a mixture A; (2) adding a solution S2 into the mixture A to extract the protein for another time, collecting all the substances so as to obtain a mixture B, wherein the volume of the solution S2 is equal to the volume of the solution S1 in the step (1); (3) subjecting the mixture B to a centrifugation treatment, collecting the supernate, adding trichloroacetic acid into the supernate to generate precipitate, and filtering so as to obtain the total protein from soybean seeds; wherein the solution S1 and the solution S2 are dedicated reagents. The soybean seed protein obtained through the method mentioned above has a better quality and higher purity. 900 protein spots can be obtained when the soybean seed protein is subjected to dimensional electrophoresis analysis, and some proteins with a low molecular weight and low abundance are clear and visible. 363 protein spots can be verified through mass spectrum, and the number of the verified protein spots is far more than that of other methods which has been reported. The method has a significant application value for research on plant proteomics.

Description

technical field [0001] The invention relates to a method for extracting soybean seed total protein and special reagents thereof. Background technique [0002] Soybean is an important economic crop in my country and the world. Its seed oil is an important source of edible oil for human and animal nutrition and food processing industry. Soybean oil consumption accounts for 31% of the world's edible oil. At the same time, soybean seeds are also rich in protein and are the main source of plant protein in the world. The identification of soybean seeds expressing whole protein is an important reference for further analyzing the mechanism of protein and oil accumulation in soybean seeds. [0003] Protein is the product of gene expression, and it is also the main executor and embodiment of many physiological functions. With the rise of functional genomics, proteomics has become one of the focuses of life science research. The purpose of proteomics research is to isolate and identi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/30C07K1/14
Inventor 曲乐庆徐秀苹
Owner INST OF BOTANY CHINESE ACAD OF SCI