problem whether a protein is located to the nucleus - please give some advice (Apr/01/2005 )
hi,everybody
I met a problem in my recent experiment. I want to test whether a protein can be located into the cell nucleus. I fused the protein to the C-terminal of EGFP and transformed into the CHO cell. The fluorescence microscope show that the fusion protein was distributed in both the cytosol and the nucleus. My protein is about 450 AA. With the EGFP about 260 AA, the Mw of fusion protein will reach > 70kd. Some paper say that there is upper size limit of ~50kd for a macromolecule to be able to diffuse through
the NPC into the nucleus. So can I reach a conclusion that my protein is located into the nucleus through a specific mechanism but not diffused into the nucleus. Please give some advice. And do you meet such an example?
This is all from memory but here goes:
Localization into the nucleus is determined by a nuclear localization signal. Now, I am not sure whether it is on the mRNA or on the protein primary sequence (my bet is on the primary protein sequence).
If you can identify the NLS on your protein, you can then take it out of your cDNA, do your CFP signal localization again and all you should see is CFP in the cytoplasm.
It is very much possible that your protein is shuttled back and forth between cytoplasm and nucleus.
OH, and another thing, try using a confocal microscope instead of an epifluorescence microscope. Ordinary fluorescence microsope pick up light in 3D, specifically, on top of the nucleus. It may look like the signal is in both, but actually you're just picking up the CFP in the cytoplasm above the nucleus.
You can also use a nuclear counterstain like DAPI.
Good luck!
Localization into the nucleus is determined by a nuclear localization signal. Now, I am not sure whether it is on the mRNA or on the protein primary sequence (my bet is on the primary protein sequence).
If you can identify the NLS on your protein, you can then take it out of your cDNA, do your CFP signal localization again and all you should see is CFP in the cytoplasm.
It is very much possible that your protein is shuttled back and forth between cytoplasm and nucleus.
OH, and another thing, try using a confocal microscope instead of an epifluorescence microscope. Ordinary fluorescence microsope pick up light in 3D, specifically, on top of the nucleus. It may look like the signal is in both, but actually you're just picking up the CFP in the cytoplasm above the nucleus.
You can also use a nuclear counterstain like DAPI.
Good luck!
Thanks for your suggestion.
Here we do not have the confocal microscope. The epifluorescence microscope is my only choice. Indeed, the confocal microscope can give me a more clear result.
Through sequence anlysis, I find no typical NLS on my protein. But I think that protein can be imported into the nucleus if they can combine with other proteins that have the NLS. I find such a example in a paper which is phosducin. Most part of this protein is in the cytoplasm, while another small part is in the nucleus with the help of another protein. (not through the NLS recognization)
Of course, to isolate the nucleus protein and do Western Blot can also give a very persuasive result. But I do not have the antibody. (Too expensive to afford, this experiment is not in plan and with no fund support)
Maybe you can isolate the nucleus (cell disruption and differential centrifugation), disrupt its contents and do spectrophotometry to read CFP fluorescence. Compare that against a non-transfected CHO cell culture. I guess you can do the same for the cytoplasmic fraction but I know you are more interested in nuclear localization.
Although I don't know how much of a sample you need for that to reach the detection threshold of a spectrophotometer.
It's just an idea. Because you already have a tagged version of your protein, try to get the most out of it.
See if that will work out.