Preparation method for procambarus clarkii ovarian tissue total protein sample suitable for dimensional electrophoresis

A technology of Procambarus clarkii and ovarian tissue, which is applied in the field of preparation of total protein samples of Procambarus clarkii ovarian tissue, can solve problems such as the preparation method of total protein samples of Procambarus clarkii ovarian tissue, and facilitate mass spectrometry analysis Identification, high reproducibility, fast and reliable results of the method

Inactive Publication Date: 2012-02-22
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there is no sample preparation method for total protein of Procambarus clarkii ovaries suitable for two-dimensional electrophoresis

Method used

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  • Preparation method for procambarus clarkii ovarian tissue total protein sample suitable for dimensional electrophoresis
  • Preparation method for procambarus clarkii ovarian tissue total protein sample suitable for dimensional electrophoresis

Examples

Experimental program
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Embodiment 1

[0028] 1. Preparation of total protein samples of Procambarus clarkii ovaries

[0029] (1) Weigh 500 mg of fresh ovary tissue of Procambarus clarkii in the developing stage, put it in a mortar pre-cooled at -20°C, cut it into pieces, grind it into powder with liquid nitrogen, and transfer it to a 15 mL centrifuge tube;

[0030] (2) Add 5 times the volume of pre-cooled solution A (which contains 0.02mmol / L dithiothreitol and a final volume concentration of 20% trichloroacetic acid) using acetone as a solvent in the centrifuge tube of step (1) , mix well, place at -20°C for 2h, centrifuge at -20°C, 12000r / min for 20min, and collect the precipitate;

[0031] (3) Add 2 mL of ice-water mixture solution B (containing 0.02 mmol / L dithiothreitol) pre-cooled to 0 °C to the precipitate in step (2), mix well, and Place at -20°C for 2h, centrifuge at -20°C, 12000r / min for 20min, and collect the precipitate. Use the pre-cooled solution B with acetone as the solvent to wash the precipitat...

Embodiment 2

[0042] 1. Preparation of total protein samples of Procambarus clarkii ovaries

[0043] (1) Weigh 500 mg of fresh ovarian tissue of Procambarus clarkii at the mature stage, put it in a mortar pre-cooled at -20°C, cut it into pieces, grind it into powder with liquid nitrogen, and transfer it to a 15 mL centrifuge tube;

[0044] (2) Add 6 times the volume of pre-cooled solution A (which contains 0.03mmol / L dithiothreitol and a final concentration of 20% trichloroacetic acid) with acetone as a solvent in the centrifuge tube of step (1), Mix well, place at -20°C for 4h, centrifuge at -10°C, 15000r / min for 20min, and collect the precipitate;

[0045] (3) Add 2 mL of ice-water mixture to the precipitation in step (2) and pre-cool to 0°C solution B (containing 0.02 mmol / L dithiothreitol) in acetone, mix thoroughly, and Place at 20°C for 3h, centrifuge at -10°C, 15000r / min for 20min, and collect the precipitate. Use the solution B with acetone as the solvent, which is pre-cooled to 0...

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Abstract

The invention discloses a preparation method for a procambarus clarkii ovarian tissue total protein sample suitable for dimensional electrophoresis. The method comprises the following steps: fully grinding ovarian tissue by using the liquid nitrogen method; precipitating proteins by using the trichloroacetic acid-acetone method, that is, precipitating the proteins by using a precooled acetone solution containing dithiothreitol and trichloroacetic acid and rinsing precipitate with a precooled acetone solution containing dithiothreitol a plurality of times; adding a sample lysate to prepare the high quality procambarus clarkii ovarian tissue total protein sample suitable for dimensional electrophoresis. The preparation method for the sample suitable for dimensional electrophoresis is rapid, reliable, simple and practicable, enables a dimensional electrophoresis pattern with high quality and good experimental repeatability to be obtained and subsequent biological mass spectrometric analysis and identification of the proteins to be convenient, and has a great practical application value.

Description

technical field [0001] The invention relates to a method for preparing a total protein sample of the ovary tissue of Procambarus clarkii, in particular to a method for preparing a sample of the total protein of the ovary tissue of Procambarus clarkii which is suitable for two-dimensional electrophoresis. Background technique [0002] Since the artificial cultivation of Procambarus clarkii was carried out in my country at the beginning of this century, its breeding scale has expanded year by year, covering an area of ​​more than 3 million mu, with an annual output of more than 500,000 tons. It has become an important emerging aquaculture resource for freshwater shrimp in my country. With the increase of domestic consumption and processing and export quantity, the number of wild resources of Procambarus clarkii has decreased significantly. Therefore, increasing the production of artificial breeding to meet the demand of the market has become the common goal and goal of the majo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/34G01N27/26C07K1/30
Inventor 水燕周鑫赵朝阳徐增洪
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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