how to insure that a protein is nuclear or not?

[Dima Klenchin]

:Hi everybody,
:
:My prot display strong NLS but immunocytochemystry failed to detect it
:in the nucleus.
:How could i cheeck whether it's because it doesn't enter the nucleus  or
:because its epitope become inaccessible when in the nucleus?
:

If you have polyclonals, then the epitope idea is extremely unlikey and 
if in immunocytochemistry your get good signal from somewhere 
else but not nucleus, then it is fair to say your protein is not 
nuclear. To make sure, dissolve cells in 1% triton - nuclei do not 
dissolve under these conditions, retain most nuclear proteins, and 
sediment at low speed. If you don't get a signal on Western from 
nuclei, then you have your answer. 

        - Dima