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Protein extraction and electrophoresis method facilitating efficient analysis of protein map on expression quantity differential protein

A protein and electrophoresis technology, applied in the field of protein spectrum engineering, can solve the problem of insufficient separation of proteins

Inactive Publication Date: 2015-04-01
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many kinds of Aspergillus niger proteins, and the physical and chemical properties of some proteins are relatively close. The existing protein electrophoresis technology cannot fully separate the proteins of Aspergillus niger.

Method used

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  • Protein extraction and electrophoresis method facilitating efficient analysis of protein map on expression quantity differential protein
  • Protein extraction and electrophoresis method facilitating efficient analysis of protein map on expression quantity differential protein
  • Protein extraction and electrophoresis method facilitating efficient analysis of protein map on expression quantity differential protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Extraction, electrophoresis, scanning and mass spectrometry analysis of protein

[0033] (1) Protein extraction

[0034] 1) Collect extracellular protein by centrifugation: 5000g of Aspergillus niger liquid fermentation broth is collected by centrifugation to collect mycelium, take 4g of mycelium and wash it with 10mL distilled water repeatedly for 6 times, and mix the liquid of washing mycelia and the liquid collected by centrifugation to collect extracellular protein. protein.

[0035] 2) Crush the cells with a low-temperature high-pressure pump to extract intracellular proteins: ①Preparation of low-temperature wall-breaking solution: 20g of absolute ethanol, 5g of trichloroacetic acid, 5g of acetic acid, 0.02g of cetylpyridine, 0.03g of sodium lauryl sulfate, distilled water Dilute to 1L and pre-cool at -20°C for 24h. ②Put 4g of the washed mycelia into 20mL of pre-cooled low-temperature wall-breaking solution, and then add it to a high-pressure homogenize...

Embodiment 2

[0053] (1) Strain and culture medium

[0054] Aspergillus niger was purchased from ATCC, the strain number is ATCC10582.

[0055] Aspergillus niger induced liquid fermentation medium: 5.0g of corn cob or rice straw alkali hydrolyzate, 0.08g of ammonium sulfate, 0.075g of peptone, 1g of glucose, 0.03g of yeast extract, 0.5g of potassium dihydrogen phosphate, 0.001g of calcium chloride, Magnesium sulfate 0.001g, ferrous sulfate 0.271mg, manganese sulfate 0.16g, cobalt chloride 0.36g, calcium chloride 0.08g, distilled water to 100mL, sterilized at 115°C for 20min. Among them, the preparation of the alkali hydrolyzate of corncob or rice straw is as follows: 100g of corncob or rice straw is crushed to 60 mesh, 100g of sodium hydroxide, 1000mL of distilled water, hydrolyzed at 30°C for 24h, after the hydrolysis, rinse with distilled water until neutral, and 60°C Dry for 72h to constant weight.

[0056] Aspergillus niger non-induced liquid fermentation medium: ammonium sulfate 0.08...

Embodiment 3

[0065] (1) bacterial strain and culture medium: with embodiment 2.

[0066] (2) Aspergillus niger liquid fermentation: with embodiment 2.

[0067] (3) Extraction of protein

[0068] 1) Collect extracellular protein by centrifugation: Same as Example 1.

[0069] 2) High-pressure pump crushing cells to extract intracellular protein: Take 4g of washed mycelia and put them into distilled water, and add them to a high-pressure homogenizer (German APV-2000-4) to homogenize and crush cells, and the crushing pressure is 1000bar.

[0070] 3) Concentration and freeze-drying of the extracted protein: same as in Example 1.

[0071] 4) cracking and centrifugation: with embodiment 1.

[0072] (4) Electrophoresis, scanning and mass spectrometry analysis: Same as in Example 1, there are 1022 Western blot spots, and there are 7 protein types with differential expression (see Table 2).

[0073] Table 2

[0074]

[0075]

[0076] From the difference between Examples 1 and 2, it can be...

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Abstract

The invention discloses a protein extraction and electrophoresis method facilitating efficient analysis of a protein map on expression quantity differential protein, and belongs to the field of protein map engineering. Protein of aspergillus niger can be efficiently extracted and protein spots can be fully separated with the protection extraction and electrophoresis method. Aspergillus niger is broken by low-temperature wall-breaking liquid for protein extraction, and the formula of the low-temperature wall-breaking liquid is as follows: 20g of absolute ethyl alcohol, 5g of trichloroacetic acid, 5g of acetic acid, 0.02g of cetylpyridinium, 0.03g of lauryl sodium sulfate and distilled water with a constant volume of 1L. The wall-breaking liquid can increase the extraction ratio of protein. The electrophoresis method is three-dimensional electrophoresis which contributes to protein extraction of aspergillus niger and differentiation of protein expressions.

Description

technical field [0001] The invention belongs to the field of protein spectrum engineering, and in particular relates to a protein extraction and electrophoresis method for efficiently analyzing proteins with differential expression in protein spectrum. Background technique [0002] Cellulose is the most abundant renewable resource on the earth. The cellulose produced by photosynthesis on the earth reaches about 10 billion tons every year. The use of cellulose to produce bioenergy is the key to alleviating the energy crisis and realizing the sustainable development of human beings. [0003] The key to cellulose utilization is to degrade cellulose into fermentable sugar-glucose. The degradation of cellulose requires the joint action of exonuclease, endonuclease and glucosidase. Among them, endonuclease degrades long fragments of cellulose into dimers, which is a key step in cellulose degradation. Degrading cellulose with cellulase has the characteristics of green, mild condi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/72
Inventor 薛栋升
Owner HUBEI UNIV OF TECH
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