Mitochondria from S. cerevisiae cells in early stat phase were isolated using a percoll gradient. As far as I know it is expected that one brownish band will appear in the gradient. However, there were two close fractions, which were tested for mitochondrial proteins (positive for both) and for ER proteins (negative for both).
I cannot find any information regarding this observation and am puzzled. Should I keep them separated or is this quite normal and everyone mixes them together?
Thank you all.
The lower band might represent intact mitochondria whereas the upper band could either contain mitochondria with compromised membrane integrity (i. e. membrane ruptures, loss of outer mitochondrial membrane) or mitochondria-associated mebranes (MAMs). To distinguish between the two possibilities I would need to see a picture of the bands in the gradient.
During isolation some mitochondria are damaged. This is unavoidable. I would recommend to only use the lower band for further analysis.
Best regards, Christian
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