Dear all,
I have a protein which full length contain 146 amino acid, and its first 14 amino acid is predicted as nuclear localization signal. After excluding 14 amino acid, I found it is localized in cytoplasm. However the full length protein does not give any signal about its localization. I repeated several time this experiment but I could not solve. I am doing PEG mediated transformation method.
If anyone could help me in this regard.
Could you try treating the cells with inhibitors such as Leptomycin B or GTP-ase inhibitors to determine whether the cytoplasmic localisation is a result of nuclear exclusion versus active nuclear export?
Thanks for your response. Probably I did not get your point, I have a construct fused with GFP and I use this one to transfect Arabidopsis protoplast. Did you mean I have to treat protoplast with inhibitor. Could you little bit clarify again. Thanks.
Hi Parvin,
Not working with Arabidopsis, it is a bit difficult to give answers that are going to necessarily be coherent. Simply - many years ago working with GFP-fused mammalian proteins that featured both putative nuclear import sequences and putative nuclear export sequences, we demonstrated that deletion of the former resulted in relocalisation of the protein to the cytoplasm, but then had to prove whether this was an active process, or simply that the mutated protein was unable to enter the nucleus. Treating mammalian cells with leptomycin B (an export blocker) was sufficient to result in accumulation of the protein within the nucleus again even in the absence of the NLS, proving that it could enter the nucleus passively and was subsequently exported actively in a dynamic shuttle mechanism.
You say that you have a 14aa sequence that is predicted to be a Nuclear Localisation sequence. When it is deleted your protein is cytoplasmic. My question is whether the protein is actively exported (which would be inhibited by LepB, suggesting that there is a Nuclear Export Sequence to identify and then mutate), or if it is simply prevented from entering the nucleus without the predicted NLS.
Pubmed suggests that LepB does work as an export blocker in Arabidopsis (0.1mg/ml for 2 h), so it may be worth trialling this inhibitor for your studies, and to then examine the sequence for potential Export motifs (these have been identified in other Arabidopsis proteins).
Hello
Thank you very much. I understand clearly now. I will try to follow your advice.
If you have any further questions, please do not hesitate to come back. That's what we are here for as fellow scientists!
Best wishes, and best of luck with your experiment.
Hi Parvin, I don't know if this helps, but your expressed protein might also be degraded by intracellular proteasomes.
Hi Steingrimur ,
So how can we know that the protein is degraded ? or anything that can stop this degradation.
Take cell lysates from the cells that are expressing the GFP fusion and resolve them by SDS-PAGE, western blot for GFP to ensure the protein is the correct molecular weight. If the protein is < the predicted mW then the protein is being degraded. If the mW is correct, then the protein is intact.
Hi Pervin, there are inexpensive cell permeable proteasome inhibitors available"
http://www.sigmaaldrich.com/catalog/product/mm/539160?lang=en®ion=US
Add it to the cells and see if GFP increases?
If you have the full length sequence of the protein, try to predict it using some prediction tools.
https://en.wikipedia.org/wiki/Protein_subcellular_localization_prediction#Tools
Hi there,
Do you mean you don't get any measurable signal with your full length protein or you get uniform fluorescence in the cell?
I might sound silly but if the former how sure are you about the construct you made for the full length protein expression?
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