Hi Oscar

Check the protein carefully....first the clone sequence....you have to make sure there are no deletions in the sequence....sometimes the tages do change the properties of the recomninant proteins

bye

HI Oscar,

It seems that there might be a problem either in cloning or in expression.. so you can try other cloning vectors also, moreover as stated by Mr. Ajay, tagging a protein sometimes changes the protein conformation, folding and activity, so do check that . if possible can you please check your protein tag by WB in the cell lysate.. (it will tell you whether your clone is really expressing in side the cell r not)....also i am not sure but did you see cell death after transfecting your construct.. ??

Hi Ajay, I already sequenced the clone and I did not find any deletions or mutations in the sequence obtained from NCBI. Debasish Paul idea of checking the protein by WB would be a good idea, although I can see it is expressed inside the cell because I can see the tetR binding spot, but it will help me in checking whether the entire fusion protein is being expressed properly. No, I didn't see cell death after tranfection, or at least not more than normal. Anyway, thank you very much!

Did you see EYFP? Is your fusion protein localized to nucleus? You can also put a C-term his- or flag-tag to the fusion protein to see if the C-term tag is expressed correctly. Or if you have a prSet7 antibody, you can directly test if your TetR-ETFP-prSet7 is correctly expressed. If expression is not a problem, the N-term fusion may indeed affecting the function of prSet7. You will then need to remove the tag or design a C-term tag.