Mitochondrial Fractionation

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Scarnati14
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Mitochondrial Fractionation

 Does anyone have a good mito fractionation protocol for yeast?

_scar

heehawmcduff
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I've done it with human lung

I've done it with human lung cells but never with yeast.  I used Pierce (Thermo) as the kit supplier so they may be able to advise if they have a solution for yeast mitochondrial extraction.

Scarnati14
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 Thanks anyway yeah I am

 Thanks anyway yeah I am actually trying to establish a general lab protocol for my laboratory that I work in. I am actually having a problem with getting some nuclear "contamination" in my mito fraction! Very frustrating.
-Scar

fissfusion10
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have you read Hartwig et al.

have you read Hartwig et al. 2009 or Hornig-Do et al., 2009 there may be something there, certainly about nuclear contamination.  They report about 70% purity.  Using a kit, I isolated intack mitochondria and then cytospun them onto slides, found nuclei as well as nice blue ribbon and smear staining /DAPI staining, as well as mitochondria stain. Interestingly, when I lysed the same isolated intake mitochondria and ran on gel/western, lysate did not stain positive for  nuclear marker, but was positive for mitochondria protein.

I also thought about using mytotracker green (live cells), then sorting out using flow