I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated from the GFP tag, the others remain tagged with GFP. I have run the same experiment with transiently transfected cells of the same type and the same plasmid remains tagged to the GFP in that western. I am not sure why the GFP tag is removed in the stably transfected cells vs. the transiently transfected cells.
You're talking about insertional mutagenesis. Any thing can happen with it. Try again, best done with a retrovirus.
Do you want to say that the protein you are detecting on the Western Blot in one case does not correspond in molecular mass to the fusion protein but to the mass of free GFP? This would not be unusual, many fusion proteins are unstable and get degraded, either in vivo or during protein isolation. Have a look to the protein sequence, maybe you can the distance between your protein and GFP bigger by inserting a linker. Or smaller, by removing some aa in between. Best, Jürgen
If you put the GFP tag in C-terminal, check if you have a start codon (ATG) in the sequence of your GFP tag. If yes, remove it and directly fuse your GFP with your protein by inserting a linker.
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