I have expressed EGFP-N1 plasmid (1ug) in MCF-7 cells. Upon taking the confocal images under 40x oil I have seen the expression all over the cell? What is the possible reason for that? Can anyone suggest me the related literature regarding that?
Hi Puneeta, why would you expect anything different? You plasmid expresses GFP at very high levels, without any specific localization signal. Thus, you will see GFP fluorescence in all cellular compartments. Please clarify, if I misunderstood you question.
Andrea Galli, yeah that is my point. If it doesn't has any specific localization signal then why it is distributed to everywhere. I was expecting to see the fluorescence signal just in the cytoplasm and not in the nucleus. EGFP doesn't have any NLS (nuclear localization signal) then why it is localizing to nucleus as well and giving fluorescence signal in all over the cell.
Puneeta Bathla, when you express EGFP alone it will localize everywhere, including the nucleus. If you insert a NLS, it will localize ONLY in the nucleus, but the absence of a NLS does not prevent the protein from going to the nucleus.
If you need cytoplasm-only localization, you can for example insert a mitochondrial targeting sequence in your plasmid.
You can have a look at this paper where they use different localization signals to detect viral infections. You can see that when the EGFP has no signals attached, it is detected in the whole cell.
Hope that helps.
Andrea Galli , Thanks. Your input on this topic is quite enlightening. So, the reason of EGFP localizing to everywhere in the cells in the absence of a particular localization signal sequence is simple diffusion or anything else?
Hi Puneeta Bathla , yes I would guess it's simple diffusion since the protein has no specific signals on it. That would be my guess, but I have no specific knowledge on this particular aspect.
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