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Hexagrammos otakii egg xanthogen western blot kit as well as preparation method, detection method and application thereof

A technology of immunoblotting and original protein, which is applied in measurement devices, biological tests, material testing products, etc., can solve problems such as toxic effects, biological hazards, carcinogenicity, etc., and achieve the effects of simple operation, increased concentration, and improved concentration and purity.

Inactive Publication Date: 2015-01-28
青岛诺亚生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, my country has not seen the research and development of POPs biomonitoring technology in the marine environment.

Method used

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  • Hexagrammos otakii egg xanthogen western blot kit as well as preparation method, detection method and application thereof
  • Hexagrammos otakii egg xanthogen western blot kit as well as preparation method, detection method and application thereof
  • Hexagrammos otakii egg xanthogen western blot kit as well as preparation method, detection method and application thereof

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Experimental program
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Embodiment 1

[0024] An immunoblotting kit for detecting vitellogenin of Otaki six-line fish, comprising a box body containing: 1) 2 PVDF membranes; 2) horseradish peroxidase-labeled goat anti-rabbit secondary antibody 1 piece; 3) 1 piece each of blocking solution, washing solution and color developing solution, the washing solution is TBST; the blocking solution is TBST containing 5% skimmed milk powder; the color developing solution contains 0.06% (m / V) 3 '-Diaminobenzidine 10mM Tris-HCl; It is characterized in that the box also contains: 4) 1 pure Otaki hexaline vitellogenin; 5) Otaki hexaline vitellogen polyclonal antibody 1 branch.

[0025] The preparation method of the above kit includes the following two parts:

[0026] (1) Preparation method of pure vitellogenin of Otaki sylvestris

[0027] Intramuscular injection 17 β -Estradiol (17 β -estradiol, E 2 ) To induce vitellogenin in Otaki fish. One week after the injection, blood was taken, centrifuged at 5000 g for 10 minutes at 4°C, and t...

Embodiment 2

[0038] The method of using the kit of the present invention to qualitatively detect the vitellogenin of Otaki sylvestris specifically includes the following steps:

[0039] 1) Perform denaturing polyacrylamide gel electrophoresis after diluting the pure vitellogenin of Otaki hexalin and the sample to be tested in the kit. The sample to be tested includes Otaki hexalin plasma, body surface Mucus, liver tissue and hepatocyte culture fluid;

[0040] 2) Transfer the protein on the electrophoresis gel to the PVDF membrane;

[0041] 3) Block the non-specific binding sites of the PVDF membrane with blocking solution, incubate overnight at 4°C, and discard the blocking solution;

[0042] 4) Add rabbit anti-Otaki vitellogenin polyclonal antibody (1:1000) diluted with blocking solution, incubate with shaking at room temperature, discard the solution, and wash the PVDF membrane with washing solution 5 times;

[0043] 5) Add horseradish peroxidase labeled goat anti-rabbit secondary antibody (1:200...

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Abstract

The invention discloses a hexagrammos otakii egg xanthogen western blot kit as well as a preparation method, a detection method and application thereof. The kit comprises a pure hexagrammos otakii egg xanthogen western blot product and an anti-rabbit hexagrammos otakii egg xanthogen western blot polyclonal antibody. The preparation method comprises the following steps: firstly, purifying egg xanthogen western blot from 17 beta-estradiol induced plasma of hexagrammos otakii through ammonium sulfate precipitation and SephadexG-200 filtration chromatography, and purifying through HitrapProteinG chromatography, thereby obtaining the anti-rabbit hexagrammos otakii egg xanthogen western blot polyclonal antibody. The kit disclosed by the invention can be used for screening ocean environmental estrogen substances, and capable of sensitively, conveniently and quantitatively detecting egg xanthogen western blot in blood, body surface mucus, liver tissue and a liver cell culture fluid, the lowest detection limit is 50ngmL<-1>, and an important tool is provided for detection on endocrine disrupter within offshore areas of China.

Description

technical field [0001] The invention belongs to the field of ecological detection, and in particular relates to an immunoblotting kit for detecting vitellogenin of Otaki six line fish, a preparation method and application thereof. Background technique [0002] Persistent organic pollutants (Persistent Organic Pollutants, POPs) are difficult to decompose in the environment, can exist in the environment for a long time, can migrate and spread on a global scale through various transmission routes, and accumulate and amplify in organisms through the food chain , a class of organic pollutants that have toxic effects on the human body and the environment. These pollutants do not exist in nature themselves, but are the products of the human industrial revolution. The hazards of persistent organic pollutants to humans and the environment have already become a global problem. In order to solve this problem, the United Nations Environment Program (UNEP) and the Swedish government co-c...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/531G01N33/689G01N33/743
Inventor 王骏王松张铁军冷凯良苗钧魁姜勇罗忻吴振兴
Owner 青岛诺亚生物科技有限责任公司
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