how to insure that a protein is nuclear or not?

[Frank O. Fackelmayer]

Denis Dupuy wrote:

> 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?

Hi Denis,
Try a biochemical cell fractionation. There are several protocols around to do that, but in your case it will be quite simple by purifying nuclei and using
western blot to see if it there or in some of the other fractions (cytoplasmic supernatant, wash...).

Frank