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Extraction separation method for soil intracellular protein

A protein separation and separation method technology, which is applied in the fields of peptide preparation, chemical instruments and methods, organic chemistry, etc., can solve the problems of interfering with the extraction of intracellular protein in soil, and achieve the effect of clear protein spots.

Inactive Publication Date: 2011-06-15
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the soil is an extremely complex "black box", and there are many interfering substances, which seriously interfere with the extraction of soil intracellular proteins. So far, there is no effective method for the extraction of soil intracellular proteins. Therefore, a fast and efficient method has been developed. The method of soil intracellular protein extraction plays an extremely important role in the study of environmental metaproteomics

Method used

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  • Extraction separation method for soil intracellular protein
  • Extraction separation method for soil intracellular protein

Examples

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Effect test

Embodiment 1

[0040] Weigh 1 g of rice soil and add 5 mL of 0.05 mol / L sodium citrate buffer into a 10 mL centrifuge tube. Shake well on a vortex shaker for 3 hours. After shaking, place the centrifuge tube in precooling to 4 o Centrifuge at 15,000 rpm for 25 min in a centrifuge in C, and discard the supernatant. Add 5mL sodium citrate buffer solution to the centrifuge tube and shake it in a vortex shaker for 10min, then pre-cool to 4 o Centrifuge at 15,000 rpm for 25 min in a centrifuge in C, discard the supernatant, and repeat 2 times. Add 5 mL of SDS buffer into the centrifuge tube, and place it in a vortex shaker for 1 hour. After shaking, the supernatant was passed through a 0.45 μm filter membrane and transferred to a new 10 mL centrifuge tube, 2 mL of Tris-saturated phenol (pH 8.0) was added, placed in a vortex shaker for 30 min, and then allowed to stand for 30 min. Then pre-cool the centrifuge tube to 4 o C centrifuge at 15000rpm for 25min, take the lower layer of phenol liqui...

Embodiment 2

[0047] Take 0.8g of sugarcane soil, add 0.05mol / L, pH8.0 sodium citrate buffer 4mL, oscillate in a vortex shaker for 2.5 hours, centrifuge at 15000rpm for 25min, discard the supernatant; take the precipitate, add 0.05mol / L, pH8.0 sodium citrate buffer 4mL, shake in a vortex shaker for 10min, then centrifuge at 15000rpm for 25min, discard the supernatant, repeat twice; take the precipitate, add 4mL of SDS buffer, shake in a vortex shaker for 50min; After the end, centrifuge at 15000rpm for 25min, collect the supernatant, and pass through a 0.45μm microporous membrane, take the filtrate and add 2mL of Tris-saturated phenol with a pH of 8.0, place it in a vortex shaker for 25min, let it stand for 20min, and then 15000rpm Centrifuge for 25 minutes, remove the lower phenol phase, add 5 times the volume and pre-cool to 4 o 0.1mol / L methanol-ammonium acetate solution of C, placed at -20 o Precipitate under C condition overnight, then centrifuge at 15000rpm for 25min; discard superna...

Embodiment 3

[0053] Take 1.5g of tobacco soil, add 0.05mol / L, pH8.0 sodium citrate buffer 6mL, shake in a vortex shaker for 4 hours, centrifuge at 15000rpm for 25min, discard the supernatant; take the precipitate, add 0.05mol / L, pH8.0 sodium citrate buffer 4-6mL and shake in a vortex shaker for 10-20min, then centrifuge at 15000rpm for 25min, discard the supernatant, repeat twice; take the precipitate, add 6mL of SDS buffer, put in a vortex shaker Shake for 80 minutes; after shaking, centrifuge at 15,000 rpm for 25 minutes, collect the supernatant, and pass through a 0.45 μm microporous membrane, take the filtrate and add 3 mL of Tris-saturated phenol with a pH of 8.0, place it in a vortex shaker for 40 minutes, and let it stand 50min, then centrifuge at 15000rpm for 25min, remove the lower phenol phase, add 5 times the volume and pre-cool to 4 o 0.1mol / L methanol-ammonium acetate solution of C, placed at -20 o Precipitate under C condition overnight, then centrifuge at 15000rpm for 25min...

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Abstract

The invention provides an extraction separation method for soil intracellular protein, which comprises the following steps: extracting with a sodium citrate extracting solution to remove soil extracellular protein; extracting and dissolving soil intracellular protein with an SDS (Sodium Dodecyl Sulfate) extracting solution, carrying out further extraction on the intracellular protein with Tris-saturated phenol, methanol-ammonium acetate and the like, and carrying out dielectrophoresis separation. The SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) detection indicates thatstripes and protein points of the extracted soil intracellular protein are clear, and the extraction separation method for soil intracellular protein is provided for the first time in the world.

Description

technical field [0001] The invention relates to a method for extracting and separating soil intracellular proteins, belonging to the field of biotechnology. Background technique [0002] As a very important part of the ecosystem, the soil microbial community plays an important role in the growth of plants, energy flow in the ecosystem, material cycle, degradation and detoxification of environmental pollutants, etc. In recent years, some new technologies for studying the diversity and interactions of microbial communities have emerged, and environmental metaproteomics technology is one of them. In addition to studying the microbial population in the environment, this method can also conduct in-depth analysis of the function of the microbial community. Although there are few metaproteomic studies that have been carried out, this technology has initially demonstrated its powerful functions: through metaproteomic research, the composition of microbial communities can be linked ...

Claims

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Application Information

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IPC IPC(8): C07K1/36
Inventor 王海斌张志兴林文雄何海斌吴林坤俞振明
Owner FUJIAN AGRI & FORESTRY UNIV
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