Localization of GFP - (Jan/06/2006 )
Hi, I am using pEGFP-N1 vector to check the localization of my fusion protein.
First of all, I would like to ask whether GFP is supposed to be cytoplasmic protein?( for me, I have thought so.)
I have noticed that the transfection with control pEGFP-N1 seems to exist in everywhere in the cells.
Is that such a case with those who had used it? or does the result varies with the type of cells we are using?
If it does exist everywhere in the cells regardless of the types of cells, do you think that it is valid experiment to show the localization of the fusion protein since the control GFP protein has no specific localization? (afterall, i couldn't explain why this particular GFP protein behaves like that)
Sorry for bombardment of questions. I will appreciate if you could help me.
what you're seeing is normal. If you use GFP as such, it will localize to the cytoplasm and nucleus. The GFP has no nuclear localization signal, but due to passive diffusion, it will be present in the nucleus as well. As for the reliability of the localization of your GFP fusion protein, that's hard to tell, but it's generally assumed that the GFP does not affect the localization, although the only way to be really sure is to look at the localization of the endogenous protein. In the absence of specific antibodies, you should fuse GFP both Nterm and Cterm to see whether there is a difference or use a different tag. If all these results are consistent, there is a big chance that will also be the normal localization of the protein.
I hope this has answered some of your questions.
Thanks! Now i feel so much better and had some ideas in mind!