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Urine exosome extraction kit based on combination of PEG precipitation method and SEC column method and application

A technology of exosomes and precipitation method, which is applied in the direction of instruments, measuring devices, scientific instruments, etc., can solve the problems that are not suitable for the processing of a large number of samples, the concentration of recovered exosomes is low, and the extraction effect is not ideal, so as to achieve low cost, Low cost, effect of improving purity

Pending Publication Date: 2022-04-26
HANGZHOU YORK BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The SEC column method is only suitable for a small amount of sample processing, such as extracting limited exosomes from a few milliliters of blood, and is not suitable for processing a large number of samples, such as directly extracting exosomes from a large number of urine samples.
Moreover, the larger the sample volume processed by the SEC column method, the more column fillers are required, the longer it takes, the concentration of recovered exosomes is low, and the extraction effect is not ideal.
The patent "Extracellular Vesicle Separation and Enrichment Method Based on Size-Exclusion Chromatography and Ultrafiltration Technology" is a method combining ultrafiltration and SEC column method, but this method uses SEC column method combined with ultrafiltration method to concentrate treatment and greatly improves The material cost is reduced, and the ultrafiltration tube is limited to only handle a small amount of urine samples

Method used

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  • Urine exosome extraction kit based on combination of PEG precipitation method and SEC column method and application
  • Urine exosome extraction kit based on combination of PEG precipitation method and SEC column method and application
  • Urine exosome extraction kit based on combination of PEG precipitation method and SEC column method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: As shown in the figure, exosome preparation:

[0052] A urine exosome extraction kit and application based on the combination of PEG precipitation method and SEC column method, comprising the following steps:

[0053] (1) 50ml of fresh morning urine was centrifuged at 180×g for 10min at 4°C, and the bottom sediment after centrifugation was discarded to obtain the urine supernatant.

[0054] (2) Centrifuge the urine supernatant obtained in (1) at 4° C., 1550×g for 20 min, discard the sediment at the bottom after centrifugation, and obtain the urine supernatant.

[0055] (3) Put the urine supernatant obtained in (2) into a freezer at -80°C for 30 minutes. After thawing at room temperature, repeat the centrifugation step in (2), centrifuge at 1550×g for 10 minutes at 4°C, discard After centrifugation, the bottom was precipitated, and the urine supernatant was obtained.

[0056] (4) Transfer the urine supernatant obtained in (3) to a new centrifuge tube. The b...

Embodiment 2

[0062] Example 2: Exosome identification:

[0063] A urine exosome extraction kit and application based on the combination of PEG precipitation method and SEC column method. The extracted exosomes are identified by western blot method and ZetaView nanoparticle tracking analyzer.

[0064] figure 2 The western blot method verified that the urine exosomes of three different individuals obtained by the PEG precipitation method, HSP70, TSG101, CD9 and CD63 are human exosome marker proteins.

[0065] image 3 The urine exosomes obtained by the PEG precipitation method were diluted 1000 times on the machine, and the ZetaView nanoparticle tracking analyzer was used for NTA detection. The detection concentration on the machine is 4.0×10 7 particles / ml, the original exosome concentration was 4.0×10 10 particles / ml.

[0066] Figure 4 The western blot method repeatedly verified that the urine exosomes obtained by combining the PEG precipitation method with the SEC column 2B, HSP70...

Embodiment 3

[0074] Example 3: Evaluation of the effect of exosome isolation and purification:

[0075] A urine exosome extraction kit based on the combination of PEG precipitation method and SEC column method and its application, the separation and purification of exosomes, and the identification of the effect of removing impurity proteins.

[0076] Figure 12 The mobile phase of the SEC column was tested for BCA. From left to right, the empty column volume, the exosome collection area, and the miscellaneous protein area. Exosomes are eluted through the SEC column before impurities such as proteins to achieve exosome purification.

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Abstract

The invention relates to a kit and application, in particular to a urine exosome extraction kit based on combination of a PEG precipitation method and an SEC column method and application. It is found for the first time that a large amount of urine crystals and other impurities can be separated out through freezing and thawing of urine, and the purity of subsequent exosome extraction is improved through low-speed centrifugation removal. According to the method, the protein concentration is detected through the BCA at the flow-through liquid sampling point, the empty column volume and a miscellaneous protein area flowing down behind the exosome are successfully avoided, and the purity of the extracted exosome is further improved.

Description

technical field [0001] The present invention relates to a kit and application, in particular to a urine exosome extraction kit and application based on the combination of PEG precipitation method and SEC column method. Background technique [0002] Exosomes are tiny membrane vesicles that can be secreted by most cells and have a lipid bilayer membrane structure with a diameter of about 30-150nm. Exosomes are naturally present in bodily fluids, including blood, saliva, urine, and breast milk. Exosomes are membranous vesicles secreted by living cells derived from late endosomes (also known as multivesicular bodies). Exosome contents: 1) Nucleic acid: rRNA, miRNA, cirRNA, lncRNA, mRNA, etc. 2) Protein: such as tubulin, actin, heat shock protein, membrane protein such as tetraspanin (CD9, CD63, CD81, CD82), integrin, annexin, etc. 3) Lipids: sphingomyelin, phosphatidylethanolamine, etc. [0003] PEG precipitation method or SEC column method is also widely used in the field of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 梁颖孔繁平
Owner HANGZHOU YORK BIOTECH CO LTD
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