I already have a GFP-tagged nuceolar-binding protein and I wish to visualise this with respect to the nuclear membrane. What is the most common nuclear membrane protein that people tag?
Nuclear lamins (intermediate filaments just underneath the inner nuclear membrane) have been very popular for GFP fusions to decorate the nuclear enveloppe from the inside, they produce nice pictures and you will find ample literature on this. If you just want to highlight the nuclear enveloppe, a soluble secretory GFP-HDEL (signal peptide-GFP-HDEL) will also illuminate it nicely. In both cases you need weak promoters to avoid non-sepcific fluorescence elsewhere.
Thank you very much for your response. Would it not be possible simply to tag the endogenous gene and thus use the endogenous promoter?
Julia
Yes, if you use the endogenous lamin promoter you can be almost certain that expression will not be too high, but you still need to make a stable integration of the hybrid gene into the genome, rather than multicopy expression from a 2mu plasmid. Otherwise you might still have unspecific labelling. I can send you GFP-HDEL or GFP-calnexin fusions, the first will label the lumen of the ER and nuclear enveloppe, the second will label the ER membrane and the outer nuclear envelope membrane. Lamins will highlight the inner nuclear membrane. All three should be very distinct from the nucleolus. :)
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