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Method Article
We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.
Materials and methods
Yeast strains and growth conditions
The wild-type strain BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) was grown in rich YEPD medium (1% yeast extract, 2% peptone, 2% glucose). Cells were cultured at 30°C with rotational shaking at 200 rpm in Erlenmeyer flasks at a "flask volume/medium volume" ratio of 5:1.
Isolation of crude mitochondrial fraction
**All subsequent steps should be carried out at 4°C or on ice. Suspensions of spheroplasts should be handled gently using pipettes with cut tips to avoid breaking organelles.**
**Although this suspension is enriched in mitochondria, it also contains other organelles such as the endoplasmic reticulum (microsomes), Golgi, and vacuoles. To get pure mitochondria, this crude mitochondrial fraction can be subjected to further fractionation, as described below.**
Purification of mitochondria devoid of contamination by other organelles
Reagents
This method enables the high-yield isolation of pure mitochondria from yeast cells. Mitochondria purified by this method are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. The described method yields mitochondria that are suitable for cell-free reconstitution of essential mitochondrial processes. These highly pure mitochondria can also be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome...
The authors have nothing to disclose.
This work was supported by grants from the CIHR and the NSERC of Canada. V.I.T. is a CIHR New Investigator and Concordia University Research Chair in Genomics, Cell Biology and Aging.
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