sub-cellular localization and fixation of cells

Michael Moser moser at U.WASHINGTON.EDU
Wed Sep 20 20:35:52 EST 1995


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.
Sincerely,

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





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