Hi all,
Simple question, maybe complex answer - is it OK to perform sub-cellular mitochondrial fractionation on cultured cells (293T etc) that have been harvested then *frozen* as pellets? Just a little worried the freezing could lyse the mitochondrial membranes - I think it's OK for yeast and animal tissues from what I can see, but mammalian cells are a little more fragile?. I generally freeze at -80C, 10e7 cell pellets of a range of cell lines - easier to prep altogether than straight after harvesting...
Context - trying to examine mitochondrial localisation of proteins, rather than assessing enzyme activity. So need clean mitochondria (and ideally resulting clean cytoplasmic/remaining material free of mitochondrial contamination); not necessarily active high activity enzymes.
Thanks!
Dear Christopher,
for your purposes I think that isolating mitochondria from frozen cells might be OK. But... There are always plenty of "buts". I mention some of them'
1. Mitochondria will be definitely unstable and might swell during the isolation procedure.; 2. Frozen cells and mitochondria even at -80 undergo rather rapid and strong free radical oxidation. I have tested this by comparing the activities of several enzymes. This means that electrophoretic properties might be different.
One of the solutions cab be to add to the freezing medium 0.25-mM dibucain, which stabilizes membranes/ In its presence even frozen overnight mitochondria preserve respiratory control and respiration.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology