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Difference between Ab-stained and GFP-tagged samples

Yann ABRAHAM yabraham at curie.fr
Mon Dec 1 11:42:57 EST 1997

In article (Dans l'article) <v01530501b09b5f89d1fa@[]>,
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,


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

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