Extraction and purification method of high-activity high-purity mitochondrial

A purification method, mitochondrial technology, applied in the field of biotechnology and biomedicine, can solve the problems of poor mitochondrial activity, experimental failure, low mitochondrial purity, etc.

Pending Publication Date: 2020-10-23
FOURTH MILITARY MEDICAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the mitochondrial separation methods used in scientific research are mainly sucrose density gradient centrifugation or commercial mitochondrial separation kits. The former requires ultra-high-speed centrifuges, which are not available in general laboratories, and the steps are cumbersome and time-consuming. The obtained mitochondrial activity Poor, although the latter greatly reduces the extraction time, but the purity of the extracted mitochondria is low, and often contains other cellular components, such as glycogen, cell debris, etc.
Often lead to experimental failure or unstable results, poor repeatability

Method used

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  • Extraction and purification method of high-activity high-purity mitochondrial
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  • Extraction and purification method of high-activity high-purity mitochondrial

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The mitochondrial extraction and purification method of mouse liver, the specific steps are as follows:

[0036] S1, take fresh mouse liver, add mitochondrial and cytoplasmic protein preparation reagents, grind 30 times with an ice-bath glass homogenizer with a tight gap, and prepare tissue homogenate; the quality of the animal tissue or animal cells: mitochondrial / cytoplasmic separation The solution volume is equal to 0.1g: 1ml;

[0037] S2, transfer the homogenate to a 2ml Eppenduf tube, centrifuge at 1000g at 4°C for 5min, discard the precipitate, and keep the supernatant;

[0038] S3, transfer the supernatant to another 2ml Eppenduf tube, centrifuge at 10,000g at 4°C for 8min, retain the precipitate, which is crude mitochondria, resuspend the precipitate with 500uL IB solution, record it as solution A, and set aside;

[0039] S4, take 500uL ice-bathed 15% Percoll solution in a 1.5mL EP tube, slowly add the A solution onto the 15% Percoll solution, centrifuge at 210...

Embodiment 2

[0042] Mitochondria Extraction and Purification of Primary Bone Marrow Macrophages

[0043] S1, take about 5 million primary bone marrow macrophages, add 1ml of mitochondrial and cytoplasmic protein preparation reagents, and repeatedly blow and suck 30 times with a 1ml syringe to prepare cell homogenate;

[0044] S2, transfer the homogenate to a 2ml Eppenduf tube, centrifuge at 1000g at 4°C for 5min, discard the precipitate, and keep the supernatant;

[0045] S3, transfer the supernatant to another 2ml Eppenduf tube, centrifuge at 10,000g at 4°C for 8min, retain the precipitate, which is crude mitochondria, resuspend the precipitate with 500uL IB solution, record it as solution A, and set aside;

[0046] S4, take 500uL ice-bathed 15% Percoll solution in a 1.5mL EP tube, slowly add the A solution onto the 15% Percoll solution, centrifuge at 21000g at 4°C for 8min, discard the supernatant, and keep the precipitate;

[0047] S5, add 800ul IB solution to the precipitate of S4, cent...

Embodiment 3

[0049] Mitochondria Extraction and Purification of HepG2 Cells

[0050] S1, take about 5 million HepG2 cells, add 1ml of mitochondrial and cytoplasmic protein preparation reagent, and repeatedly blow and suck 30 times with a 1ml syringe to prepare cell homogenate;

[0051] S2, transfer the homogenate to a 2ml Eppenduf tube, centrifuge at 1000g at 4°C for 5min, discard the precipitate, and keep the supernatant;

[0052] S3, transfer the supernatant to another 2ml Eppenduf tube, centrifuge at 10,000g at 4°C for 8min, retain the precipitate, which is crude mitochondria, resuspend the precipitate with 500uL IB solution, record it as solution A, and set aside;

[0053] S4, take 500uL ice-bathed 15% Percoll solution in a 1.5mLEP tube, slowly add the A solution on top of the 15% Percoll solution, centrifuge at 21000g at 4°C for 8min, discard the supernatant, and keep the precipitate;

[0054] S5, add 800ul IB solution to the precipitate of S4, centrifuge at 15,000g at 4°C for 5min, ...

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Abstract

The invention relates to the field of a biological technique and biological medical care, in particular to an extraction and purification method of high-activity high-purity mitochondrial. The methodcomprises the following steps of taking animal tissue or cells, adding a mitochondrial and plasmosin preparing reagent, and performing homogenization to obtain homogenate; performing centrifugation onthe homogenate, removing precipitate, and reserving supernatant; performing centrifugation on the supernatant, reserving precipitate to obtain coarse extracted mitochondrial, and performing re-suspension on the coarse extracted mitochondrial with an IB solution to obtain a solution recorded as a solution A; mixing an ice bath 15% Percoll solution with the solution A in the volume ratio being 1 to1, performing centrifugation, discarding supernatant, and reserving precipitate; and adding the precipitate to the IB solution, performing centrifugation, removing supernatant, reserving precipitate,cleaning the reserved precipitate, and removing the Percoll solution to obtain purified mitochondrial. Compared with mitochondrial extracted through a commercial reagent kit sold in the market, the mitochondrial obtained by the method is higher in purity and higher in activity, and can be used for scientific experiment research.

Description

technical field [0001] The invention relates to the fields of biotechnology and biomedicine, in particular to a method for extracting and purifying mitochondria with high activity and high purity. Background technique [0002] Mitochondria are important organelles in eukaryotic cells. They are the site of cellular energy metabolism and oxygen metabolism. They participate in oxidative phosphorylation, tricarboxylic acid cycle, store calcium ions, etc., and play a key role in the process of cell survival and death. Mitochondria are among the most sensitive organelles to various damages. After cells are damaged, changes in the structure and function of mitochondria, such as abnormalities in membrane potential, respiratory chain, protein localization, cytochrome C, oxygen free radicals, etc., can directly or indirectly cause cell necrosis or apoptosis, and eventually cause various diseases. occur. Mitochondrial gene, structural and functional abnormalities have been found to b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
CPCC12N5/0645C12N5/067C12N2509/00
Inventor 张坤冯源赵吉倩武胜昔王亚周郇宇
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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