What could be the cause for this GFP expression pattern...? - (Mar/17/2010 )
When I transfect my cells with empty GFP vector, fluorescence pattern is citoplasmatic and nuclear, as it should be.
But when I transfect cells with my construct (a GFP tagged version at c-ter of my protein of interest, which I suspect to interact with a membrane receptor and is predicted to be citoplasmatic in in silico studies), it seems to have a bright punctate pattern (perhaps compatible with localization at membrane microdomains) along with a quite intense fluorescene in citoplasm and nucleus!!!
Should I suspect an artifactual localization of my fusion protein? Why I found it in the nucleus?
Any suggestion on how I must continue these experiments is welcome.
GFP is a quite large tag and, despite its common use, is often not a good model for this sort of thing - it commonly inhibits normal functions of tagged proteins through steric hindrance. You will probably be better off determining the localisation of your protein of interest with immunofluorescence, using an un-tagged vector for transfection if need be.
It's tricky, you have to try both N-link and C-link to see which one works better.
Regarding your punctuate pattern, it may be due to the wildtype has more expression than the mutant