Method for gathering phosphorylated proteins and method for detecting phosphorylated proteins

A phosphorylated protein and enrichment technology, which is applied in measurement devices, preparation of test samples, instruments, etc., can solve the problems of enrichment effect and selection limitation, and achieve the effect of wide applicability, high sensitivity, and avoidance of denaturation.

Inactive Publication Date: 2019-06-21
INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the specificity of the antibody-antigen reaction, the enrichment effect and selection of this method are limited by the selected antibody

Method used

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  • Method for gathering phosphorylated proteins and method for detecting phosphorylated proteins
  • Method for gathering phosphorylated proteins and method for detecting phosphorylated proteins
  • Method for gathering phosphorylated proteins and method for detecting phosphorylated proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Enrichment of phosphorylated proteins

[0059] 1) Take 5.21gFeCl 3 ·6H 2 O, 4.22 g FeSO 4 ·7H 2 O, add 250mL of pure water to dissolve completely, add 850μL of concentrated hydrochloric acid, ultrasonically deoxygenate for 30min, add ammonia water, adjust the pH to greater than 10, stir at 1000rpm at 80°C for 40min, stop stirring, let stand for 60min, and use magnetic separation to separate the precipitate from the reaction medium , the separated magnetic fluid is fully washed with pure water, washed 4 times with absolute ethanol, and the obtained magnetic fluid is dispersed in 500mL of absolute ethanol;

[0060] 2) Add 250mL of pure water to the magnetic fluid, ultrasonically disperse in the three-neck flask for 10min, add 38mL of ammonia water, 50mL of ethyl orthosilicate to the dispersion, stir at 60°C for 3h, stop the reaction, fully wash with ethanol and vacuum dry overnight , get Fe 3 o 4 @SiO 2 magnetic particles;

[0061]3) Get 3mL glacial acetic acid an...

Embodiment 2

[0066] Detection of phosphorylated proteins

[0067] Take 2 μL of the casein enriched solution obtained in Example 1 and mix with 6 μL of mesonic acid, take 2 μL of the mixed solution and spot it on a stainless steel target plate, and use MALDI-TOF-MS to measure it. The test results are as follows: figure 2 shown.

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Abstract

The invention provides a method for gathering phosphorylated proteins and method for detecting phosphorylated proteins and belongs to the technical field of phosphorylated protein detection. The method comprises steps that a phosphorylated protein sample and the buffer solution are mixed with a chitosan functionalized magnetic material, after magnetic separation, a phosphorylated protein-enrichedmagnetic material is obtained; the phosphorylated protein-enriched magnetic material is mixed with the acid solution, after magnetic separation, the phosphorylated protein-enriched solution is obtained; a core of the chitosan functionalized magnetic material is Fe3O4, and an outer layer of Fe3O4 is sequentially wrapped by silica and chitosan polymeric materials. The method is advantaged in that the method can overcome limitations of a traditional immunoprecipitation enrichment method for antibody and has wide applicability.

Description

technical field [0001] The invention relates to the technical field of protein enrichment detection, in particular to a phosphorylated protein enrichment method and a phosphorylated protein detection method. Background technique [0002] The post-translational modification of proteins plays a very important role in the life process, and the modification of the same protein at different sites may correspond to different functions. Phosphorylation modification of protein is one of the important modification methods in organisms, and it plays a biological "on / off" effect on the cellular functions of many organisms. Therefore, it is very necessary to study phosphorylated proteins and establish high-sensitivity detection technology for analyzing the complex and diverse physiological and pathological processes of living organisms. [0003] After the protein is phosphorylated and modified, its molecular mass will change accordingly, and the molecular mass of the protein can be acc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/40G01N27/64
Inventor 周焕英高志贤梁俊王蕊王永辉
Owner INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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