Does anyone have a good mito fractionation protocol for yeast?
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.
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.
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