In article (Dans l'article) <v01530501b09b5f89d1fa@[194.214.93.66]>,
marqueze at jean-roche.univ-mrs.fr (Beatrice Marqueze) wrote (écrivait) :
> 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.
First, I used the mAB against GFP in western blotting : I diluted the
antibody 2000 times, and in one case where my extract was heavily
concentrated, I obtained a good signal with a 2nd antibody linked with
alkalin phosphatase. However, it might be best to use ECL revelation,
because with poorly concentrated extracts, you're not able to detect them
in the conditon I describe.
Second, we observed an overlapping signal between GFP fluorescence and
antibody staining when we tried this. May be the non overlapping signal you
observe is a local concentration of GFP being destroyed. It happens from
time to time when the overexpression is too strong for the cell.
Good luck,
Yann
--
Yann ABRAHAM
Institut Curie
Laboratoire "Biologie du cycle cellulaire et de la Motilité"
UMR 144 - C.N.R.S.
26 rue d'Ulm
75231 Paris cedex 5
