cellular localization

[meggie82]

Dear members,
I have some discrepancy. Western blot shows nuclear localization whereas immunocytochemistry – cytoplasmic localization of the protein.
We know that antibody is working, it was checked in different cell types, where it shows staining of the nucleus. It was also used in Western blot with another cell type overexpressing our protein and it worked as well in nuclear fraction.
For fractionation we use a Qiagen kit, but somehow I have a feeling that it must be technical problem of correct separation of cellular fractions. We suspect also that the protein can be attached to nuclear envelope and nuclear location can be linked to the developmental stage of the cells.
Is there any good protocol to separate soluble and insoluble proteins from nuclear fraction?

Cheers,
Meg

[JackBean]

do you have some controls on your Western (to make sure, your fractions are pure)?