Dear colleagues!

We are trying to isolate mitochondria from conifer plants. The regular Percoll step gradient isn't working due to the percularities of the material, so we want to try mixed Percoll-Sucrose gradient.

What should be the sucrose concentration of the steps? The percoll works better with steps 50%, 25% and 18%. Wanted to try this:

1. 18% Percoll 0.25 M Sucrose

2. 25% Percoll 0.5 M Sucrose

3. 50% Percoll 1.2 M Sucrose

But this cannot be done due to excess sucrose that won't fit in volumes (for 10 ml step we need to add 5 ml Percoll, 2 ml of Medium x5 (which is already pretty dense) and somehow add 3.2 g of sucrose - seemes unreal).

I wonder if we need to try to preserve the osmolarity of the steps and equalize it with another sugar (Mannitol?)? What do you think?