GFP, both in the nucleus and in the cytoplasm - transfection (Dec/11/2007 )
hi all,
I am a newbee in transfection. I have transfected my muller glia cell line with pEGFP-C3 vector. GFP is present both in the nucleus and in the cytoplasm in my transfected cells. I was thinking that GFP alone would be only in the cytoplasm because there is no reason for it to go into nucleus. In some papers, I have seen that GFP is located both in the nucleus and cytoplasm in transfected COS-7 or HeLa cell lines. Is this the case?
Are you observing GFP in everywhere in your transfected cells??
Attached is the picture of my GFP-transfected cells.
Thank you all...
GFP as such will normally locate both in the cytoplasm and in the nucleus, your results are good!
If you use confocal, you can get clearcut results. Otherwise, you could misidentify the egfp that is in the cytoplasm located just above or below the nuclei.
The staining look fine.
This is normal.
Thank you guys,
genehunter, we don't have confocal, I am using inverted microscope 
why do you think GFP enters nucleus?
When I looked my cells transfected with EGFP vector, I saw some cells have them exclusive in cytoplasm, some are mixed, and if these cells are rounded either in replication or dying, you have this nuclear-localized pattern, which could be true nuclear or just happens to be about or below the nucleus, therefore fluorescence microscopy under high mag or a confocol will be the best to address this question, is it or it is not nuclear localized?
Apparantly, you are not the only one who raised the question. Check this link for a work on this subject. http://www.ncbi.nlm.nih.gov/sites/entrez?c...Cisrctnupittlib
I hope it helps.
GFP will randomly enter because it is a small protein and can diffuse in. This is why you see it sometimes and not, usually proteins smaller then 60 kDa can enter the nucleus by simple diffusion. This is why tagging a putative NLS or NES is a great control, if you find it in the cytoplasm all the time you know your NES is working because GFP CAN enter the nucleus and vice-versa.
I am a newbee in transfection. I have transfected my muller glia cell line with pEGFP-C3 vector. GFP is present both in the nucleus and in the cytoplasm in my transfected cells. I was thinking that GFP alone would be only in the cytoplasm because there is no reason for it to go into nucleus. In some papers, I have seen that GFP is located both in the nucleus and cytoplasm in transfected COS-7 or HeLa cell lines. Is this the case?
Are you observing GFP in everywhere in your transfected cells??
Attached is the picture of my GFP-transfected cells.
Thank you all...
EGFP is not enriched in the nucleus if there is no NLS;