Method for efficiently extracting low-abundance protein of soybean seeds

Abstract

The invention belongs to the technical field of extraction and identification of crop seed components, and particularly relates to a method for efficiently extracting low-abundance protein of soybean seeds. The method comprises the following steps: S1, mixing dried and mature soybean seeds with isopropanol with the volume concentration of 30-60% according to a ratio of (100-120mg): 1ml, and then performing oscillation extraction for 1-1.5 hours to obtain a protein extracting solution; s2, centrifuging the protein extracting solution for 8-10 minutes, mixing supernate with precooled acetone according to the volume ratio of 15: 10, sufficiently whirling, and standing the mixed solution at the temperature of -20 - -15 DEG C; and S3, after 10-12 hours, centrifuging the mixed solution at the temperature of 4 DEG C at the speed of 6000-8000 r / min for 18-20 minutes, and naturally drying the centrifugal precipitate for 30 minutes to obtain the low-abundance protein of the soybean seeds. According to the extraction method, the low-abundance protein can be preferentially enriched, the interference of the high-abundance protein of the soybean seeds on the low-abundance protein is effectively eliminated, and a high-quality protein sample with few impurities, a clear electrophoresis band, high resolution and biological activity is provided for research of soybean proteomics and metabonomics.

Description

technical field

[0001] The invention belongs to the technical field of extracting and identifying components of crop seeds, and in particular relates to a method for efficiently extracting low-abundance proteins from soybean seeds. Background technique

[0002] With the deepening of molecular biology, structural biology, genomics and other research, people realize that it is not enough to elucidate the phenomenon and essence of life activities only by genome sequence. Many researchers have shifted the focus of research in the field of crop science from genes to proteins, making proteins an important research object for revealing life activity phenomena and molecular biological mechanisms. The first step in protein research is to separate and purify the target protein from complex macromolecular mixtures to obtain high-purity, biologically active targets. Therefore, efficient purification techniques and means are an important basis and key step for protein research. [0003...

Examples

Embodiment 1

[0022] Extraction of Low Abundance Proteins from Soybean Seeds

[0023] Total protein extracts were prepared by extracting protein from dried mature soybean seeds with isopropanol: Grind the seeds in liquid nitrogen using a pre-cooled mortar and pestle, weigh 500 mg of soybean seed powder, and place it in a 5 mL of 40% volume concentration isopropanol in a centrifuge tube. Protein extraction was performed on an orbital shaker at a speed of 18,500r / min for 1h. The protein extract was centrifuged at 13,000 r / min for 10 min at 4°C, and the supernatant was taken and placed in a clean 15 mL glass tube. 10 mL of pre-cooled acetone was added to the supernatant, and vortexed thoroughly, then the protein extract was placed at -20° C. overnight (12 h). The next day, the sample was centrifuged at 4°C for 20 min at a speed of 8,000 r / min, and the centrifuged precipitate was dried at room temperature for 30 min. The precipitate was resuspended in 0.5mL lysis buffer [the lysis buffer con...

Embodiment 2

[0025] Ability of different concentrations of isopropanol to preferentially dissolve low-abundance soybean seed protein

[0026] The extraction method of the present embodiment is the same as that of the embodiment 1, the difference is that eight groups whose volume concentrations of isopropanol are respectively 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80% are set.

[0027] In order to compare the ability of different concentrations of isopropanol solutions to preferentially dissolve low-abundance soybean seed proteins, it was analyzed by SDS-polyacrylamide gel electrophoresis. The gel concentration was 15%. Proteins were visualized after overnight staining with Coomassie Blue G-250. Protein extracts with 10% and 20% concentration of isopropanol are rich in proteins, including high-abundance soybean seed storage proteins such as β-conglycinin and glycinin ( figure 1 , lane 1 and lane 2). When the concentration of isopropanol was 30%, 40%, 50% and 60%, the extraction of high-ab...

Embodiment 3

[0029] Two-dimensional gel electrophoresis and in-gel digestion and mass spectrometric identification of low molecular weight protein spots

[0030] 1. Two-dimensional gel electrophoresis

[0031] In order to further analyze and identify low-abundance soybean seed proteins by two-dimensional gel electrophoresis, soybean seed proteins were extracted again with four different volume concentrations of isopropanol (30%, 40%, 50% and 60%), and the centrifuged precipitate was placed in Dry at room temperature for 30min. The precipitate was resuspended in 0.5mL lysis buffer [the lysis buffer contained 9mol / L urea, 1% CHAPS by volume, 0.5% IPG buffer by volume and 1% by mass dithiothreitol], and vortexed until the precipitate was completely dissolved to obtain a protein solution.

[0032] In the first dimension, proteins were separated according to their isoelectric points, and a 13 cm, pH 4-7 linear IPG gel strip from GE Healthcare was used for isoelectric focusing electrophoresis....

technical field

[0001] The invention belongs to the technical field of extracting and identifying components of crop seeds, and in particular relates to a method for efficiently extracting low-abundance proteins from soybean seeds. Background technique

[0002] With the deepening of molecular biology, structural biology, genomics and other research, people realize that it is not enough to elucidate the phenomenon and essence of life activities only by genome sequence. Many researchers have shifted the focus of research in the field of crop science from genes to proteins, making proteins an important research object for revealing life activity phenomena and molecular biological mechanisms. The first step in protein research is to separate and purify the target protein from complex macromolecular mixtures to obtain high-purity, biologically active targets. Therefore, efficient purification techniques and means are an important basis and key step for protein research. [0003...

Examples

Embodiment 1

[0022] Extraction of Low Abundance Proteins from Soybean Seeds

[0023] Total protein extracts were prepared by extracting protein from dried mature soybean seeds with isopropanol: Grind the seeds in liquid nitrogen using a pre-cooled mortar and pestle, weigh 500 mg of soybean seed powder, and place it in a 5 mL of 40% volume concentration isopropanol in a centrifuge tube. Protein extraction was performed on an orbital shaker at a speed of 18,500r / min for 1h. The protein extract was centrifuged at 13,000 r / min for 10 min at 4°C, and the supernatant was taken and placed in a clean 15 mL glass tube. 10 mL of pre-cooled acetone was added to the supernatant, and vortexed thoroughly, then the protein extract was placed at -20° C. overnight (12 h). The next day, the sample was centrifuged at 4°C for 20 min at a speed of 8,000 r / min, and the centrifuged precipitate was dried at room temperature for 30 min. The precipitate was resuspended in 0.5mL lysis buffer [the lysis buffer con...

Embodiment 2

[0025] Ability of different concentrations of isopropanol to preferentially dissolve low-abundance soybean seed protein

[0026] The extraction method of the present embodiment is the same as that of the embodiment 1, the difference is that eight groups whose volume concentrations of isopropanol are respectively 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80% are set.

[0027] In order to compare the ability of different concentrations of isopropanol solutions to preferentially dissolve low-abundance soybean seed proteins, it was analyzed by SDS-polyacrylamide gel electrophoresis. The gel concentration was 15%. Proteins were visualized after overnight staining with Coomassie Blue G-250. Protein extracts with 10% and 20% concentration of isopropanol are rich in proteins, including high-abundance soybean seed storage proteins such as β-conglycinin and glycinin ( figure 1 , lane 1 and lane 2). When the concentration of isopropanol was 30%, 40%, 50% and 60%, the extraction of high-ab...

Embodiment 3

[0029] Two-dimensional gel electrophoresis and in-gel digestion and mass spectrometric identification of low molecular weight protein spots

[0030] 1. Two-dimensional gel electrophoresis

[0031] In order to further analyze and identify low-abundance soybean seed proteins by two-dimensional gel electrophoresis, soybean seed proteins were extracted again with four different volume concentrations of isopropanol (30%, 40%, 50% and 60%), and the centrifuged precipitate was placed in Dry at room temperature for 30min. The precipitate was resuspended in 0.5mL lysis buffer [the lysis buffer contained 9mol / L urea, 1% CHAPS by volume, 0.5% IPG buffer by volume and 1% by mass dithiothreitol], and vortexed until the precipitate was completely dissolved to obtain a protein solution.

[0032] In the first dimension, proteins were separated according to their isoelectric points, and a 13 cm, pH 4-7 linear IPG gel strip from GE Healthcare was used for isoelectric focusing electrophoresis....

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