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Zymogram analysis method for semi-cellulosome analogue

An analysis method, semi-fiber technology, applied in the analysis of materials, material analysis by electromagnetic means, measurement devices, etc., can solve the problem of non-specificity of the substrate

Inactive Publication Date: 2018-11-30
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the early stages of the technique, substrates were not specific and were used only to detect broad proteolytic activity

Method used

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  • Zymogram analysis method for semi-cellulosome analogue
  • Zymogram analysis method for semi-cellulosome analogue
  • Zymogram analysis method for semi-cellulosome analogue

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Experimental program
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Embodiment Construction

[0027] The specific implementation manners of the present invention will be further described below in conjunction with the accompanying drawings and technical solutions.

[0028] 1. Electrophoresis

[0029] Firstly, a continuous non-denaturing gradient gel with a concentration range of 2% to 15% by mass was prepared using a gradient apparatus, and the formula is shown in Table 1.

[0030] Table 1 Gel formulation

[0031]

[0032]

[0033] After gel preparation, start sample loading. The sample consists of the supernatant obtained by centrifugation from the fermentation broth of strain F16 and Loading Buffer for non-denaturing electrophoresis. Then start electrophoresis, the condition is 80V, 120min.

[0034] 2. Zymogram Analysis

[0035] After electrophoresis, put the gel into the gel containing 0.5mM MUX and 1mM CaCl 2 50mM pH 7.5 Tris-HCl buffer and incubated at 30°C for 5min. Afterwards, the gel was quickly observed under a 365nm ultraviolet light, and photogra...

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Abstract

The invention provides a zymogram analysis method for semi-cellulosome analogue and belongs to the technical field of industrial biology. The method disclosed by the invention is characterized in thatthe whole analysis process is composed of three parts: electrophoresis, zymogram analysis and proteinogram analysis. A continuous gradient non-modified gel without concentrated gel is adopted for electrophoresis; an off-on probe substrate is adopted for zymogram analysis; coomassie blue staining or silver staining is adopted for proteinogram analysis. According to the method, once electrophoresisis performed while zymogram and proteinogram information of a block of gel are collected; if multienzyme complex is generated in supernatant of microorganism fermentation liquor can be quickly and effectively judged; the zymogram analysis method is developed for screening microbial strains capable of generating cellulosome compounds and can be applied to the fields of industrial bioconversion, biological energy source, medical intermediate preparation, and the like.

Description

technical field [0001] The invention relates to a zymogram analysis method for cellulite analogues, which belongs to the field of industrial biotechnology. Background technique [0002] Zymography is an experimental technique for studying hydrolytic enzymes through substrate degradation transformations. Zymography was introduced in 1962 when Gross and Lapière developed an assay to detect collagen degradation in tadpole tissue and described matrix metalloproteinases for the first time. Through zymography analysis, it is possible to visually show that the substrate is converted into a reaction product through hydrolysis, and the biochemical reaction is marked by a detection method specific to the appearance of the reaction product or the disappearance of the substrate. Therefore, zymography techniques can be used to develop hydrolases for any substrate: proteins and peptides, oligosaccharides and polysaccharides, lipids and even nucleic acids. Any kind of biological sample c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447
CPCG01N27/447
Inventor 窦同意陈静
Owner DALIAN UNIV OF TECH
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