With most plants, it's very simple: Just isolate chloroplasts of
dark-adapted plants(i.e. at least overnight, maybe 36 hours to be on the
safe side. You'll have to experiment a little). With seedlings you
should have no problem at all, I think.
There are some plants (and mutants), however, which store <a lot> of
starch in their chloroplasts.
Since I don't know if you want thylakoid membranes or intact
chloroplasts, the answer will be complex:
For thylakoids, you can do a simple sucrose density centrifugation.
Starch has a much higher density.
To separate thylakoids from chloroplasts (spinach), I regularly used a
40% Percoll solution (in summer 42%, so play with the parameters) with
300mM Sorbit or Sucrose as osmoticum. Thylakoids will stay above this
layer, and chloroplasts sedimented (look up e.g. Heimann and Schreiber
1996, Photosynthesis Research 47: 187-197). You can carry this one step
further and put another layer of, let's say, 45 or 50% Percoll below
this. In this case, the heavier starch should pass this layer but not
the chloroplasts. This will of course rupture the chloroplast envelope,
but if the starch was not too big, the envelope should reseal itself.
You should expect some enzyme loss.
If you have further questions, you can contact me at
heimanns at bilbo.bio.purdue.edu
Sebastian Heimann
Hongwei Ji wrote:
>> I tried to isolate chloroplast from plant seedling, but there are so mucn
> starch mixed up with. Is there any good idea to deal with this?
>> Any help would be greatly appreciated. Thanks a lot!
>> H.W. Ji
