help to see whether my protein is imported to the nucleus - please give some advice (Apr/01/2005 )
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? What other experiments can help to get a clearer result?
Why not isolate the nucleus protein and do Western Blot ?
why dont u isolate nuclear and cytosolic extracts of ur cells and do western blots separately and quantify them to look at the % of protein in nucleus and cytosol ?
I don't know if it'll work (I'm a student, so I'm not so good ) but you can fuse HA epitopes to your protein sequence and detect the fusion with antibodies anti-HA by western blot or immunofluorescence
I'm a student too. Thanks for your suggestion. I will try it.
I'm from China. There is a restriction in my lab that we can not buy the reagent more than 1000 RMB. The antibody from foreign companies is mostly about 3000 RMB, too expensive that our boss is not willing to buy. I can only use the antibody produced by domestic company. It is a terrible thing to do experiment in such a lab.