Dear Peter (and others),
I have used essentially similar fixation conditions with
formaldehyde on yeast cells expressing GFP-calmodulin. I did my fixation
for 15 minutes at 4 C. Fixation seemed to reduce the overall
fluorescence, but did not affect the localization. The longer I fixed
the dimmer the fluorescence seemed to appear.
Maybe (complete conjecture) the fixed localization represents a
true localization. Over-expression of GFP-calmodulin in yeast results in
cytoplasmic staining not present when expressed at a lower level. If
GFP-cyclophilin is overexpressed in COS cells it may fill the cytoplasm.
Protein not interacting with a target my fail to be crosslinked during a
short fix and is subsequently washed away revealing the previously
obscured filamentous structure. Just an idea off the top. I welcome any
comments or criticisms, I think this could be an important question.
Mike Moser Tel: 206-543-5354
UW Department of Biochemistry FAX: 206-685-1792
Box 357350 moser at u.washington.edu
Seattle, WA 98195-7350 Make peace my beast is yeast