For what it's worth, and based on my experience with rat liver homogenates
long ago, I would not even try fractionation!

It's worth doing a very basic fractionation into nuclear/mitochondrial,
endoplasmic reticulum and high speed sup fractions, but beyond that, I
think it is very hard to get definitive assignment to Golgi, contractile
vacuole, or vehicles of the endocytic pathway. The various sorts of
gradient you can use will give broad, overlapping fractions and if you are
looking at vesicles, you will have to work hard to keep them intact.

Better to improve your tagging or antibody......

Rob


> Dear Dicty-colleagues,
>
> We are trying to figure out the subcellular localization of a membrane
> protein. Due to tagging and expression issues, we are not able to do this
> by microscopy. However,  we can detect the protein by Western blot. So we
> are wondering if it is possible to determine the localization by
> subcellular fractionation.
>
> Does anyone have a good protocol for separating different organelles in
> Dicty? Such as sucrose or percoll gradient?
>
> Many thanks in advance,
>
> Huaqing
>
> _________________________________________
>
> Prof. Dr. Huaqing Cai
> Institute of Biophysics
> Chinese Academy of Sciences
> 15 Datun Rd, 6223, Chaoyang District
> Beijing
> China
>
>