I need a protocol to isolate algae and mitochondria from my symbiont cnidarian, so that I can use theses different component for enzymatic localization studies?
these portions, mainly the mitochondrial and algal one, would goes to different analysis. so I need them in the best conditions.
Also I have the choice to use the sonication or Fast-Prep using grinding beads. in your opinion which is best??
Best Regards Colleagues
In my experience (with hard corals and zoanthids) you need to treat the algae gently if you want to get them out intact and metabolically active. Our papers (two are attached) have the method we used; also one of our papers has the method for getting rid of the symbiosome around the algae, if that is needed for your experiments. I have no experience of isolating mitochondria from animals or algae. My instincts tell me that you would want to remove the algae intact first, to avoid contamination of the animal mitochondria with algal cell contents, but, as I said, I have not tried to isolate the mitochondria.
Good luck with this interesting work,
Rosalind Hinde
A simple method have been used with good success!
Article Temperature Induced Changes in Thylakoid Membrane Thermostab...
This method will almost certainly leave host cytoplasm (including mitochondria) with the zooxanthellae. if you want the algae without the mitochondria you will need to wash them by centrifugation; several washes are needed, with the coral we used, to remove all traces of coral cytoplasm and organelles.
After isolation zooxantellae could be cultured using e.g. f/2 medium, whereby you would exclude host cytoplasm etc.
Yes, that is true as long as you realise that some Symbiodinium cannot be grown in culture and that those that can may change their properties in culture. But you do make a fair point.
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