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Wild ginseng protein extracting method suitable for dielectrophoresis

An extraction method, two-dimensional electrophoresis technology, applied in the field of wild ginseng protein extraction, to achieve the effects of reducing denaturation precipitation, improving solubility, and increasing protein content

Inactive Publication Date: 2010-12-08
BEIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, wild ginseng contains a large amount of interfering substances such as nucleic acids, polysaccharides, and polyphenols, and the protein extraction method reported in the literature cannot effectively remove these substances. Therefore, the present invention optimizes the lysate formula of the protein extraction method on this basis. , the cells were broken up effectively, and a set of protein extraction method for wild ginseng suitable for two-dimensional electrophoresis was established

Method used

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  • Wild ginseng protein extracting method suitable for dielectrophoresis
  • Wild ginseng protein extracting method suitable for dielectrophoresis
  • Wild ginseng protein extracting method suitable for dielectrophoresis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. Extracting 20-year-old wild ginseng protein and performing two-dimensional electrophoresis analysis

[0020] (1) Take fresh 20-year-old wild ginseng, grind it fully in liquid nitrogen, weigh 0.15 g of the ground powder in liquid nitrogen, and transfer it to a centrifuge tube.

[0021] (2) Add 1ml of pre-cooled acetone solution containing 0.07% β-mercaptoethanol at -20°C, mix well, let stand at -15°C for 1-2h, centrifuge at 8000rpm at 2°C for 15min, discard the supernatant, and take the precipitate

[0022] (3) Step (2) was repeated three times until the supernatant was colorless, and the precipitate was dried at room temperature for 20-30 min to obtain a crude protein extract.

[0023] (4) Dissolve wild ginseng protein extract in 450 μl of lysis buffer containing 7mol urea, 2mol thiourea, 0.02g / ml CHAPS, 20mmol / LDTT, 1% PMSF, 1% plant protease inhibitor, and place the sample in In the ice-water mixture, the ultrasonic probe was immersed in the center of the ...

Embodiment 2

[0027] Example 2. Extraction of thirty-year-old wild ginseng protein and two-dimensional electrophoresis analysis

[0028] (1) Take fresh 30-year-old wild wild ginseng, grind it fully in liquid nitrogen, weigh 0.15 g of ground powder in liquid nitrogen, and transfer it to a centrifuge tube.

[0029] (2) Add 1ml of pre-cooled acetone solution containing 0.07% β-mercaptoethanol at -20°C, mix well, let stand at -20°C for 1-2h, centrifuge at 8000rpm at 4°C for 15min, discard the supernatant, and take the precipitate

[0030] (3) Step (2) was repeated three times until the supernatant was colorless, and the precipitate was dried at room temperature for 20-30 min to obtain a crude protein extract.

[0031] (4) Dissolve wild ginseng protein extract in 450 μl of lysis buffer containing 7mol urea, 2mol thiourea, 0.02g / ml CHAPS, 20mmol / LDTT, 1% PMSF, 1% plant protease inhibitor, and place the sample in In the ice-water mixture, the ultrasonic probe was immersed in the center of the sam...

Embodiment 3

[0035] Example 3. Extracting 40-year-old wild ginseng protein and performing two-dimensional electrophoresis analysis

[0036] (1) Take fresh 40-year-old wild wild ginseng, grind it fully in liquid nitrogen, weigh 0.15 g of ground powder in liquid nitrogen, and transfer it to a centrifuge tube.

[0037] (2) Add 1ml of pre-cooled acetone solution containing 0.07% β-mercaptoethanol at -20°C, mix well, let stand at -25°C for 1-2h, centrifuge at 8000rpm for 15min at 8°C, discard the supernatant, and take the precipitate

[0038] (3) Step (2) was repeated three times until the supernatant was colorless, and the precipitate was dried at room temperature for 20-30 min to obtain a crude protein extract.

[0039] (4) Dissolve wild ginseng protein extract in 450 μl of lysis buffer containing 7mol urea, 2mol thiourea, 0.02g / ml CHAPS, 20mmol / LDTT, 1% PMSF, 1% plant protease inhibitor, and place the sample in In the ice-water mixture, the ultrasonic probe was immersed in the center of the...

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Abstract

The invention discloses a wild ginseng protein extracting method suitable for dielectrophoresis. The method comprises the following steps of: taking wild ginseng as an experimental material, fully grinding the wild ginseng in liquid nitrogen and transferring the obtained product to a centrifuge tube; adding acetone solution precooled at the temperature of -20 DEG C into the centrifuge tube, washing sample powder for 3 times, collecting sediments by centrifugation and drying the obtained product at room temperature for 20min to obtain the crude extract of the wild ginseng protein; dissolving the crude extract of the wild ginseng protein into lysis buffer, and performing ultrasonic cell disintegration and extracting the supernate by centrifugation to obtain the high-quality solution of the wild ginseng protein. The wild ginseng protein extracting method for the dielectrophoresis is simple and fast, can obtain an electrophoresis pattern having a high repeatability and resolution, is favorable for subsequent protein identification by mass spectrographic analysis and provides a technical support for the proteomic research of the wild ginseng.

Description

technical field [0001] The invention belongs to a protein extraction method, in particular to a wild ginseng protein extraction method suitable for two-dimensional electrophoresis. Background technique [0002] Wild ginseng grows naturally in virgin forests and belongs to the herbaceous plant of the Araliaceae Panax genus. As early as 2,000 years ago, the medicinal value of wild ginseng was recorded in detail in "Shen Nong's Materia Medica", and Li Shizhen called wild ginseng a divine herb in "Compendium of Materia Medica". With the development of medicine, the miraculous curative effect of wild ginseng has been recognized by more and more people. This precious ginseng is indispensable in the prescriptions of traditional Chinese medicine for treating hypertension, coronary artery sclerosis, angina pectoris, diabetes, cancer and other major diseases. However, its medicinal mechanism is still unclear. Two-dimensional electrophoresis technology is one of the most effective me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 孙立伟麻锐姜锐冯凯
Owner BEIHUA UNIV
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