A large proportion of mislocalization of my overexpressed GFP fused protein

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AllenChiu
AllenChiu's picture
A large proportion of mislocalization of my overexpressed GFP fused protein

My GFP fused protein have a large proportion of mislocalization when overexpressed by transfection.
It is supposed to localize in nucleolus,but actually in 90% of the GFP positive cell it localized in nucleus.
Do you have any experience like this?Do you have any suggestion?
Thanks very much.

Sami Tuomivaara
Sami Tuomivaara's picture
AllenChiu,

AllenChiu,

It is not unheard of that GFP causes mislocalization of proteins. There can be several potential reasons. First, the overexpression can simply overwhelm the capacity of the target compartment/site and most of the protein leaks back out. Second, fused GFP can mask the localization signal, I think the signal can be either N- or C-terminal, or the GFP can cause misfolding of the protein of interest and this way interfere with the localization.

You can try to switch the termini of protein of interest and GFP in your fusion, or use less DNA in your transfection... These come to my mind right now...

Cheers,