Geetings,
I am interested to know what experiences people have had with GFP
and GFP fusion proteins with regard to their sub-cellular localization
before and after fixation.
I have been expressing GFP-cyclophilin fusions in COS-7 cells and
have been looking at their localization. I find that following fixation
with 4% aqueous paraformaldehyde, 20 minutes at RT, the proteins show a
different sub-cellular location as compared with the live cells.
Specifically the proteins appear to have a roughly uniform distribution
throughout the cell in live cells and following fixation the cytoplasmic
portion of the fluorescence seems 1) reduced in intensity overall and 2)
associated with filamentous structures in the cytoplasm. The nuclear
fluorescence still appears uniform and dosent appear to have lost intensity.
I have also investigated MeOH fixation, 1-2 minutes at RT and 10
minutes at -20. Both of these conditions appear to cause a fairly
dramatic loss of fluorescence intensity but do not result in the apparrent
relocalization of proteins to filamentous structures as was the case with
formaldehyde.
I am thinking that the formaldehyde may cause an actual change in
localization by crosslinking these proteins selectively with other
proteins that they may have weak interactions with and that the MeOH
fixation may be denaturing GFP and causing a loss in fluorescence.
Has anyone else noticed similar phenomena or have other
interpretations of my observations? Does any one know of fixation
conditions that do not cause these problems?
Thanks in advance for any replies
Peter Belshaw
Dept. of Chemistry
Harvard University
