Membrane isolation - (Oct/17/2006 )

Hello,
I´d like isolate nuclear and ER membranes from 293 cells. Do anybody have a protocol to do this using a sucrose gradient?
Thanks in advance.
Sergio

nuclei can be pelleted (~7.500xg), for other membrane use 10-40% sucrose gradient; you have to use a swing out rotor in an ultracentrifuge; mini UC is convenient but may give not enough protein; use as much as possible protein/cells; you may have established screening assays for reference enzymes to discriminate between different enriched membrane frations (ER, Golgi, Perox., plasma membrane, vesicle etc)