Recently there's been discussion about what type of fixation works with
GFP. Perhaps these observations will help. Most of my experience with
GFP is in mammalian cells, although I have been purifying it in E.coli as
well.
Free soluble GFP diffuses quickly. Methanol fixation may permeabilize
cells enough that all the GFP leaks out of the cell before it can be
fixed. GFP fusions may diffuse less quickly and remain in place long
enough to be fixed. People having success with methanol fixation are
probably fixing GFP fusion proteins. My GFP fusions fix fine with MeOH,
whereas the cytoplasmic GFP I use for a transfection control does not fix
well in MeOH.
Paraformaldehyde or glutaraldehyde fix GFP expressing cells fine in my
hands--both GFP fusions and free GFP. What destroys GFP fluorescence is
the NH4Cl quench afterwards.
Hope this is useful,
jamie
--
Jamie White
Light Microscopy Group
Cell Biophysics Programme
European Molecular Biology Laboratory
finger jwhite at phenix.embl-heidelberg.de
