Extraction method of total protein from rumen epithelial tissue of milk cow

A technology of epithelial tissue and extraction methods, applied in the fields of peptide preparation, chemical instruments and methods, organic chemistry, etc., can solve problems such as the lag of proteome research, and achieve the effect of obvious integrity and visualization

Inactive Publication Date: 2014-02-12
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, due to the complexity of the rumen epithelial tissue, the research on the proteome related to the rumen epithelial tissue is relatively lagging behind

Method used

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  • Extraction method of total protein from rumen epithelial tissue of milk cow

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Extraction of total protein samples from rumen epithelial tissue

[0033] 1) Separate the cow rumen epithelial tissue, cut it into small pieces with a sterile scalpel, place it in a pre-cooled mortar, add liquid nitrogen and grind it to powder, and transfer the tissue powder to a centrifuge tube.

[0034] 2) Add pre-cooled 10% trichloroacetic acid in acetone solution (every 1 L of acetone contains 0.1 kg trichloroacetic acid) into the centrifuge tube, vortex and mix well, and place it overnight at -20°C. Then centrifuge at 10000×g for 15 min, discard the liquid phase, and collect the precipitate A.

[0035] 3) Add 4 times the volume of 80% cold acetone solution (each 1 L of solution contains 0.8 L of acetone and 0.2 L of water) to the tissue precipitate A, vortex fully, place at -20 ° C for 1 h, and then pass through 10000 × Centrifuge at g for 15 min, discard the liquid phase, and collect the precipitate B.

[0036] 4), lyophilize the tissue precipitate B, add an ...

Embodiment 2

[0054] 1. Extraction of total protein samples from rumen epithelial tissue

[0055] 1) Separate the cow rumen epithelial tissue, cut it into small pieces with a sterile scalpel, place it in a pre-cooled mortar, add liquid nitrogen and grind it to powder, and transfer the tissue powder to a centrifuge tube.

[0056] 2) Add pre-cooled 10% trichloroacetic acid in acetone solution 5 times the volume of tissue powder into the centrifuge tube, shake and vortex to mix, place at -20°C overnight, then centrifuge at 10000×g for 15min, discard the liquid phase fraction, collect the precipitate A.

[0057] 3) Add 5 times the volume of 80% cold acetone solution to the tissue precipitate A, vortex fully, place at -20°C for 2 hours, then centrifuge at 10,000×g for 15 minutes, discard the liquid phase, and collect the precipitate B.

[0058] 4) Freeze-dry the tissue pellet B, add an equal volume of lysate containing 8mol / L urea, 2mol / L thiourea, 4% CHAPS, 100mmol / L Triton X-100 and 65mmol / L ...

Embodiment 3

[0076] 1. Extraction of total protein samples from rumen epithelial tissue

[0077] 1) Separate the cow rumen epithelial tissue, cut it into small pieces with a sterile scalpel, place it in a pre-cooled mortar, add liquid nitrogen and grind it to powder, and transfer the tissue powder to a centrifuge tube.

[0078] 2) Add pre-cooled 10% trichloroacetic acid in acetone solution 3 times the volume of tissue powder into the centrifuge tube, shake and vortex to mix, place at -20°C overnight, then centrifuge at 10000×g for 15min, discard the liquid phase fraction, collect the precipitate A.

[0079] 3) Add 3 times the volume of 80% cold acetone solution to the tissue precipitate A, vortex fully, place at -20°C for 0.5h, then centrifuge at 10000×g for 15min, discard the liquid phase, and collect the precipitate B .

[0080] 4) Freeze-dry the tissue pellet B, add an equal volume of lysate containing 8mol / L urea, 2mol / L thiourea, 4% CHAPS, 100mmol / L Triton X-100 and 65mmol / L dithiot...

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Abstract

The invention relates to an extraction method of protein from animal tissues, in particular to an extraction method of total protein from a rumen epithelial tissue of a milk cow. The method comprises the steps of putting a rumen epithelial tissue sample of the milk cow in a precooled mortar, adding liquid nitrogen, grinding into powder, adding an acetone solution of precooled trichloroacetic acid, oscillating, eddying, uniformly mixing for overnight aging, centrifugally collecting sediment, freeze-drying the tissue sediment, adding lysate, incubating and eddying at intervals, and centrifugally collecting supernate, namely the total protein of the rumen epithelial tissue of the milk cow. For the total protein of the rumen epithelial tissue of the milk cow, extracted by the method, a total protein mixture can be separated by a two-dimensional gel electrophoresis technology according to an isoelectric point and molecular weight of the protein, the relative abundance of the protein is acquired, the isoelectric point, the molecular weight and a relative transcript level of each protein can be clearly and visually shown in a gel atlas, and the method has the advantages of more obvious integrity and visualization in comparison with the prior arts such as a high efficiency liquid chromatography separation method, an enzyme-linked immunosorbent assay and immunoblotting.

Description

technical field [0001] The invention relates to a method for extracting animal tissue protein, in particular to a method for extracting total protein of cow rumen epithelial tissue. Background technique [0002] The rumen is a unique digestive organ of ruminants, and it is the main place for microbial digestion and physical digestion. The development and function of the rumen epithelium directly affect the production performance of ruminants, and there is an academic point of view of "healthy rumen, healthy cows". [0003] The rumen epithelium is composed of stratified squamous epithelial cells, which are divided into four layers, from the serosa layer to the mucosa layer: basal layer, spinous layer, granular layer and stratum corneum. The development of proteomics research technology provides a powerful research tool for in-depth understanding of the role of rumen epithelial tissue in the digestion and absorption of dietary nutrients. Proteomics is a scientific field that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/30C07K1/28C07K1/14
Inventor 杨永新赵小伟程广龙黄冬维赵辉玲
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE ANHUI ACAD OF AGRI SCI
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