Dear colleagues!
We are currently conducting protocol adaptation for isolation of mitochondria from freshwater amphipods. The isolation medium was from the protocol of mitochondria isolation from shrimp (Sucrose 0.125M, D-Sorbitol 0.375M, KCl 0.15M, EGTA 0.001M, MOPS 0.02 M, and 0.5% of BSA), and the wash medium with the same content except for lower concentration of EGTA - 0.025 mM.
During the isolation there was very strange upper phase after the second centrifugation - bright red precipitate on the upper side of a tube. Probably some pigments.
The mitochondria precipitate was heterogenic - the most heavy part of beige color (obviously mitochondria) and the other part of orange - that time thought to be mitochondria accidently dyed with pigment.
Then we conducted Percoll gradient centrifugation (the self-forming gradient, 30% Percoll) and expected mitochondria to be in relatively low part of a gradient. On the contrary, after the centrifugation (40000 g, 60 min) the gradient contained mitochondria layer only 5 mm from the sufrace (it was 38 ml tube). Instead being cloudy, the layer seemes like some sort of aggregates.
After the washing procedure entire mitochondria fraction was bright orange.
Next we tried to measure the protein content with Bradford method and found that mitochondria, being finely dispersed throughout the media, soon starts to aggregate and form flakes.
Bradford works poor - the aliquotes that shows 100 mcg of mitochondrial protein after centrifugation were a pellets like 300-400 mcg. So I propose that the detergent in Bradford is somehow insufficient for total solubilization of mitochondria.
Furthermore, when we conducted detergent solubilization procedures (digitonin and DDM) - after the procedures the pellets of unsolubilized mitochondria were almost like before solubilization. Plus, the DDM had 3 phases instead of 2: the lower unsolubilized mitochondria pellet, solubilizate interphase and flake-like bright orange upper phase seemed to be the pigment.
Our thoughts are: in amphipods body there are some agents that results in mitochondria aggregation and subsequent partial detergent resistance. Are there some procedures that we can add to remove them?
For the isolation we used full body of amphipods. Maybe the aggregating agents were in their gastrointestinal tract.
Also, we want to try discontinious Percoll but unsure about the steps - the only paper that I found on animal mitochondria discontinious Percoll gradient centrifugation was rat brain mitochondria (Sims et al., 2008), but not sure if it is applicable for crustaceans.
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