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What could be the cause for this GFP expression pattern...?


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#1 EGF

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Posted 17 March 2010 - 07:30 AM

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.

#2 bob1

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Posted 17 March 2010 - 03:31 PM

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.

#3 Functional Screens

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Posted 18 March 2010 - 07:06 PM

It's tricky, you have to try both N-link and C-link to see which one works better.

#4 EGF

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Posted 23 March 2010 - 12:01 AM

I add some pictures for clarification. Any idea about what this punctate pattern should mean? It is not present in the mutant construct of the fusion protein...

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#5 Functional Screens

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Posted 25 March 2010 - 01:13 PM

It all depends on the protein features and how you fused them. I have done some fusions and in the pictures 4 and 5 they're just N- and C- fusion and you can see huge difference.

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#6 Functional Screens

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Posted 25 March 2010 - 01:19 PM

Regarding your punctuate pattern, it may be due to the wildtype has more expression than the mutant




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