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Mitochondria separation method

A separation method, mitochondrial technology, applied in the biological field, can solve the problems of inconvenient use, slow reaction speed, low yield, etc., and achieve the effect of protecting activity, fewer steps, and shorter time consumption

Active Publication Date: 2021-06-29
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It solves the problems of high cost, low yield, slow reaction speed, poor effect and inconvenient use of the existing technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] A method for isolating mitochondria

[0128] 1. Preparation of separation liquid:

[0129] Sucrose, EGTA, HEPES, fatty acid-free bovine serum albumin are mixed with water; In the separation liquid, sucrose final concentration 320mM, EGTA final concentration 1mM, HEPES final concentration 10mM, fatty acid-free bovine serum albumin final concentration 0.5% (g / mL);

[0130] The pH was adjusted to 7.2 using KOH.

[0131] The prepared separation solution can be stored at 4°C for 1 month and at -20°C for 6 months.

[0132] 2. Prepare ice and pre-cool the centrifuge to 4 degrees. The following steps (step 3) need to be completed on ice and kept at 0-4 degrees.

[0133] 3. Collect cells

[0134] A) Take the adherent cell culture system, place the culture system containing the cultured cells and the culture medium in a T75 culture bottle, pipette the medium and discard the culture medium, rinse with the separation solution, and discard the separation solution;

[0135] B) ...

Embodiment 2

[0152] The difference between this embodiment and Example 1 is only in step 1, in the preparation of the separation liquid: the final concentration of sucrose in the separation liquid is 278mM, the final concentration of EGTA is 0.1mM, the final concentration of HEPES is 5mM, and the final concentration of fatty acid-free bovine serum albumin is 0.5%. (g / mL)

Embodiment 3

[0154] The difference between this embodiment and embodiment 1 is only in step 1, in the preparation of separating liquid:

[0155] Mix sucrose, mannitol, EGTA, HEPES, fatty acid-free bovine serum albumin with water; the final concentration of sucrose in the separation liquid is 70mM, the final concentration of mannitol is 210mM, the final concentration of EGTA is 1mM, the final concentration of HEPES is 10mM, and the final concentration of fatty acid-free bovine serum Albumin final concentration 0.5% (g / mL).

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Abstract

The invention discloses a mitochondria separation method which comprises the following steps: 1) resuspending and culturing cells by using a separation medium to obtain a cell suspension; 2) connecting a Teflon grinder to an IKA stirrer to grind the cell suspension; 3) performing centrifugal treatment to obtain supernatant; and 4) performing centrifugal treatment on the supernatant, and collecting precipitate. The separation method is low in cost and simple to operate, time consumption is shortened, the final mitochondria yield is increased, the complete mitochondria can be obtained, the energy metabolism activity of the mitochondria can be kept, and follow-up metabolism related detection can be carried out.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a method for separating mitochondria. Background technique [0002] Mitochondria is an important organelle in cells. It is unique to eukaryotic cells and exists in most living cells. It is an important organelle responsible for energy conversion. [0003] The energy metabolism function of mitochondria, that is, oxidative phosphorylation, has become a popular target for drug development. At present, many oxidative phosphorylation inhibitors have been reported to be applicable to the treatment of various cancers, and some drugs have entered the clinical trial stage . The electron transport chain is the main structure of mitochondria for oxidative phosphorylation, which consists of five complexes. The study of the effects of drugs on the activity of these complexes is an important basis for screening oxidative phosphorylation inhibitors. Current kits for detecting the activi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2509/10C12N2509/00
Inventor 周彦妮
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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