Felix Bestvater wrote:
> we've tagged our gene of interest with two different GFPs at both
> sequence termini simultaneously in order to exclude nuclear import
> caused by passive diffusion. the complete protein should now be about
> 84 kDa and is still accumulated inside the nucleus 24h after
> transfection. according to the literature proteins with more than 68
> kDa should not be able to get into the nucleus by passive diffusion
> at all.
>> is it thinkable that the GFPs are faided in through the
> nuclear pore consecutively like pearls of a pearl necklet? does
> anyone have experience with it?
>> thank you for any help
Unfortunately you don´t tell us details about your protein of interest.
You don´t seem to like the idea that your protein is in the nucleus.
What do you mean when you say "the protein is accumulated in the
nucleus"? Is it clearly and exclusively nuclear, or is there
fluoresescence in the cytoplasm as well but the nucleus seems to be more
fluorescent? The latter is often observed for GFP alone, but results
from the bulkiness of the nucleus as compared to the optically quite
If you want to exclude diffusion of your protein into the nucleus, make
a simultaneous fusion with GFP and a big heterologous protein like
beta-Galactosidase (adding an extra 120kD). If the construct still goes
to the nucleus, it has a functional NLS that you might (or might not...)
detect by sequence analysis.