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Flammulina velutipes mitochondria extraction method

An extraction method and mitochondrial technology, applied in the field of microorganisms, can solve the problems of long time consumption, poor mitochondrial purity, mitochondrial outer membrane pollution, etc.

Active Publication Date: 2020-10-30
NANJING UNIV OF FINANCE & ECONOMICS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the poor purity of the extracted mitochondria, differential centrifugation is rarely used anymore.
The discontinuous density gradient centrifugation method is a relatively traditional extraction method. The extraction of mitochondria is relatively pure, but the operation is complicated and time-consuming, and before the mitochondria are dispersed into the suspension medium, the mitochondria will have short-term contact with the contents of the vacuoles. The outer mitochondrial membrane has the risk of being contaminated and lysed by these contents; at the same time, repeated high-speed centrifugation and high concentration of sucrose can easily cause damage to the inner mitochondrial membrane

Method used

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  • Flammulina velutipes mitochondria extraction method
  • Flammulina velutipes mitochondria extraction method
  • Flammulina velutipes mitochondria extraction method

Examples

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Effect test

Embodiment 1

[0054] Immerse the sample in mercaptoethanol solvent, soak at 32°C for 20min, centrifuge at 500×g for 3min, discard the supernatant, resuspend the pellet in sorbitol, add 35mg·g helicase -1 (according to the weight of the precipitate obtained in the above steps), incubate at 37°C for 1h; centrifuge at 500×g for 3min, discard the supernatant, wash the precipitate three times with PBS, and collect the precipitate by centrifuging at 500×g for 3min at 4°C; add 1.5 Resuspend the pellet in ml ice-cold Mito-Cyto Buffer (derived from the mitochondrial-cytoplasmic protein preparation kit purchased from Beijing Pulilai Gene Technology Co., Ltd.), and the volume ratio of the pellet mass to Mito-Cyto Buffer is 1g: 5ml, 4°C, centrifuge at 800×g for 5 minutes, collect the supernatant and centrifuge again at 800×g for 5 minutes, collect the supernatant and centrifuge again at 10,000×g for 10 minutes, the sediment at the bottom of the tube is thick mitochondria. The pellet was resuspended wit...

Embodiment 2

[0056] The steps are the same as in Example 1, except that the enzymatic hydrolysis time is 55 minutes.

Embodiment 3

[0058] The steps are the same as in Example 1, except that the enzymatic hydrolysis time is 65 minutes.

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Abstract

The invention provides a flammulina velutipes mitochondria extraction method, and belongs to the technical field of microorganisms. The flammulina velutipes mitochondria extraction method provided bythe invention comprises the following steps of: soaking a sample with a sulfhydryl compound solvent, centrifuging and discarding a supernatant to obtain a soaked sample; re-suspending the soaked sample with sorbitol to obtain a re-suspension of the soaked sample, adding helicase into the re-suspension of the soaked sample for enzymolysis, centrifuging, and discarding a supernatant to obtain an enzymatically hydrolyzed sample; resuspending the sample subjected to enzymolysis by using a Mito-Cyto Buffer, and centrifuging to collect a supernatant; and centrifuging the supernatant obtained in theprevious step, and collecting the precipitate to obtain the mitochondria. The flammulina velutipes mitochondrion extraction method provided by the invention is simple to operate and short in consumedtime, and the extracted mitochondrion is complete in structure and high in functional activity.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for extracting mitochondria of Flammulina velutipes. Background technique [0002] At present, the extraction methods of Flammulina velutipes mitochondria mainly include differential centrifugation and discontinuous density gradient centrifugation. Due to the poor purity of the extracted mitochondria, differential centrifugation is rarely used anymore. The discontinuous density gradient centrifugation method is a relatively traditional extraction method. The extraction of mitochondria is relatively pure, but the operation is complicated and time-consuming, and before the mitochondria are dispersed into the suspension medium, the mitochondria will have short-term contact with the contents of the vacuoles. The outer mitochondrial membrane has the risk of being contaminated and lysed by these contents; at the same time, repeated high-speed centrifugation...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/06C12R1/645
CPCC12N1/14C12N1/06
Inventor 马宁王鹤潼方东路胡秋辉王朝
Owner NANJING UNIV OF FINANCE & ECONOMICS
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