Method for separating exosomes from plasma on basis of immobilized metal affinity chromatography

A separation method and technology of exosomes, applied in the biological field, can solve the problems such as there are no reports on the separation and enrichment of exosomes, and achieve the effect of low cost

Pending Publication Date: 2019-12-10
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Immobilized affinity chromatography microspheres CAE-Ti 4+ -IMAC is a technical tool of IMAC, which was used to enrich phosphorylated peptides of proteins in previous research applications, and there is no application report on the isolation and enrichment of exosomes

Method used

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  • Method for separating exosomes from plasma on basis of immobilized metal affinity chromatography
  • Method for separating exosomes from plasma on basis of immobilized metal affinity chromatography
  • Method for separating exosomes from plasma on basis of immobilized metal affinity chromatography

Examples

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Embodiment 1

[0040] Example 1 Isolation and Characterization of Plasma Exosomes

[0041] 1. Method

[0042] (1) The solid-phase metal affinity chromatography weighing 4mg is a monodisperse immobilized affinity chromatography microsphere, (CAE-Ti 4+ -IMAC), and the microspheres were washed 3 times with 500 μL of phosphate buffered saline (PBS, pH 7.5), and centrifuged at 500 g for 1 min.

[0043] (2) The collected human plasma was filtered through a 0.22-micron filter to remove cell debris, dead bodies, and large vesicles, and then 100 μl was mixed with 100 μl PBS, and then added to the solution containing CAE-Ti 4+ -In a 1.5ml centrifuge tube of IMAC;

[0044] (3) Rotate and mix the centrifuge tube containing the mixture at 4°C for 10 minutes;

[0045] (4) Wash the microspheres 3 times with 500 μl of PBS, and centrifuge at 500 g for 3 min to remove non-specifically bound proteins and molecules;

[0046] (5) To extract exosome protein, take 100 μl of SDT solution (2% SDS, 0.1M DTT, 0.1M T...

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Abstract

The invention provides a method for simply and rapidly separating exosomes or exosome proteins from plasma on the basis of the immobilized metal affinity chromatography. The method at least comprisesthe following steps: 1), the exosomes are adsorbed by monodispersed immobilized affinity chromatographic microspheres CAE-Ti<4+>-IMAC; 2), the exosomes are eluted by ammonium hydroxide; and 3), phosphate buffer is immediately utilized to replace ammonium hydroxide, and damage of ammonium hydroxide to the exosomes is reduced. The collecting method has the characteristics of being low in cost, simple and fast to operate, time-saving, labor-saving and high in collecting efficiency, and besides, various kinds of subsequent molecular analysis are allowed to be performed on the exosomes with the method and include analysis for genomes, proteomes and the like. The problems of high time consumption, high cost, low purity and the like of current exosome separation and purification can be effectively solved, the feasible technical scheme is provided for research of disease plasma exosomes, and the method has good application prospects in the aspect of discovery of new exosome markers and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for simple and rapid separation of exosomes in plasma by solid-phase metal affinity chromatography. Background technique [0002] Exosomes are small membrane vesicles (30-200nm) that contain complex RNA and proteins. The exosome membrane structure is very similar to the cell membrane structure. They are composed of phospholipid bilayers with a thickness of about 5nm. The components mainly include ceramide, cholesterol, sphingolipids, and glycerophospholipids with long saturated fatty chains. The surface and interior of exosomes are rich in various proteins, and contain important biological macromolecules such as multiple nucleic acids. Different cells may secrete specific proteins and RNAs, whose main function is to transmit information between cells, and play important physiological and pathological functions in various biological states and diseases, including tumorigenes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C07K1/14
CPCC12N5/0634C07K1/145C12N2509/00
Inventor 张勇袁玉佳杨浩程惊秋
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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