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Method for separating and purifying exosomes

A technology of exosomes and miscellaneous proteins, which is applied in cell dissociation methods, biochemical equipment and methods, artificial cell constructs, etc., to achieve the effects of simple operation, stable results, and short time-consuming

Inactive Publication Date: 2018-11-23
辽宁润基生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The patent (a method for isolating and purifying different exosome subgroups) describes the use of hydroxyapatite for size exclusion chromatography for separation, which is gradient eluted with different concentrations of phosphate to obtain different fractions , to obtain exosomes of different subgroups and analyze them, but the exosome suspension obtained from different subgroups contains a large amount of impurity proteins, not pure exosomes, but only a mixed solution containing exosomes, Therefore, there is an urgent need for a method to obtain pure exosomes

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  • Method for separating and purifying exosomes
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  • Method for separating and purifying exosomes

Examples

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Effect test

Embodiment 1

[0046] Example 1 The method of isolating and purifying total exosomes from human serum and plasma:

[0047] 1) Determination of eluate concentration when isolating and purifying exosomes from samples

[0048] (1) Plasma pretreatment: thaw the frozen plasma sample (from Liaoning University of Traditional Chinese Medicine) at room temperature, centrifuge at 4°C, 3000g for 15min, discard the precipitate, and leave the supernatant to pass through a 0.22μm filter membrane to remove large cysts Bubble. Pipette 500 μL of plasma into a 1.5 mL centrifuge tube, add 125 μL of the exosome extraction reagent in the exosome extraction kit (Cat#EXORG30A-1), repeatedly blow and mix with a pipette, and incubate at 4°C for 30 min (or overnight) , and then centrifuged at 4°C and 1500g for 30min, discarded the supernatant, and then centrifuged at 4°C and 1500g for 5min to completely remove the supernatant. Add 950 μL of 10 mM NaCl to the precipitate to dissolve it completely. What is obtained ...

Embodiment 2

[0140] According to the above optimization conditions, a complete set of methods for the separation and purification of exosomes from serum and plasma to obtain relatively pure exosomes was obtained. Specific steps are as follows:

[0141] (1) Frozen plasma samples (from Liaoning University of Traditional Chinese Medicine) were thawed at room temperature, centrifuged at 3000g at 4°C for 15 minutes, discarded, and the supernatant was passed through a 0.22 μm filter membrane to remove large vesicles, and then 157 μL of plasma was absorbed Into a 1.5mL centrifuge tube, add 45 μL of the exosome extraction reagent in the exosome extraction kit (Cat#EXORG30A-1), repeatedly blow and mix with a pipette, incubate at 4°C for 30 min (or overnight), and Centrifuge at 4°C and 1500g for 30min, discard the supernatant, and then centrifuge at 4°C and 1500g for 5min to completely remove the supernatant. Add 300 μL of 10 mM NaH to the collected pellet 2 PO 4 to dissolve completely. What is ...

Embodiment 3

[0150] The method for isolating and purifying total exosomes from cell culture fluid and urine comprises the following specific steps:

[0151] (1) 10-15ml cell culture medium (the cell culture medium is respectively LM3 / BALL / Lncap / HepG 2 ) Centrifuge at 4°C, 2000rpm for 5min to remove the suspended cells, centrifuge the supernatant at 4°C, 12000g for 20min, discard the precipitate, leave the supernatant and filter through a 0.22 micron pore filter membrane to remove larger microbubbles, absorb 5ml of cell culture solution In a 15ml centrifuge tube, add 750 μL of the exosome extraction reagent in the exosome extraction kit (Cat#EXORG24B-1) to the centrifuge tube, repeatedly blow and mix with a pipette, and place at 4°C for at least 30 minutes (or overnight).

[0152] (2) Operate according to the instructions in the exosome extraction kit (Cat#EXORG24B-1), and add 300ul of 10mM NaH to the collected precipitate 2 PO 4 Obtain exosome resuspension containing contaminating prote...

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Abstract

The invention belongs to the technical field of biological detection, and relates to a method for separating and purifying exosomes. Exosome suspension containing impure protein is obtained from a sample through separation, and hydroxyapatite is used for separation and purification to obtain the pure exosomes. The method is simple and convenient to operate and low in cost, high-purity exosome particles can be obtained fast and efficiently, and downstream experiments of exosome protein analysis, exosome DNA / RNA extraction, exosome electron microscope analysis, exosome particle size analysis andthe like are convenient; the method has very good application prospects.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a method for separating and purifying exosomes. Background technique [0002] Exosomes (exosomes) are microvesicles with a plasma membrane bilayer structure containing complex proteins, lipids, nucleic acids and other active biomolecules, with a diameter of about 30-150 nm, which are mainly derived from intracellular lysozyme The multivesicular bodies formed by the invagination of body particles are released into the extracellular matrix after the fusion of the multivesicular outer membrane and the cell membrane. [0003] Exosomes are currently regarded as specifically secreted membrane vesicles, which participate in intercellular communication, immune antigen presentation, neurotransmitter transmission, lipid metabolism and cell signal transduction, and are involved in the occurrence, development and treatment of various diseases. are closely related to prognosis. S...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2509/00
Inventor 李萍赵卓张绍峰康伟苏加忱李淑君马南王绍成吕志
Owner 辽宁润基生物科技有限公司
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