Difference between Ab-stained and GFP-tagged samples

Beatrice Marqueze marqueze at jean-roche.univ-mrs.fr
Fri Nov 21 10:30:31 EST 1997


I have transfected in CHO a GFP fusion-protein (EGFP-N1 plasmid from
Clontech). I often do not see a perfect overlap between the gfp signal and
the signal obtained with an antibody against the protein fused. The overlap
is partial. I observe a very strong gfp emission of light in a sub-cellular
compartment which could be the Golgi but antibodies there do not interact
with the same intensity. Could it be that this compartment is   not well
permeable to my antibodies ? Or is the expression of the fusion  protein so
strong in that structure that the antibodies cannot access to the epitopes
? Or is it because the fluorescence of GFP is affected by the pH or
something else in this specific compartment ?

To understand this difference I would like to compare the gfp signal in my
CHO cells with the one obtained with the monoclonal antibody against GFP
from Clontech?  Does someone has already tried this experiment ? Have you
obtained perfect overlap ?
Does anybody try the Clontech monoclonal antibody against GFP in Western
blot, immunoprecipitation or immunohistochemistry. What dilutions have you
used ?

Thanks in advance for advices and comments.


..............................................................................
Beatrice Marqueze-Pouey
INSERM U 374/464, Institut Federatif Jean Roche, Faculte de Medecine-Nord,
13916 Marseille Cedex 20, France.
Tel (33) 4 91 69 88 32, Fax (33) 4 91 09 05 06,
E-mail: marqueze at jean-roche.univ-mrs.fr
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