Hello All,
I am working on the protein which is abundantly found in cytoplasm. But small fraction of it also goes to the nucleus and where it perform its nuclear function. I am interested in its nuclear function aspect in control vs treated condition. For which, I want to go for differential mass spec of that protein only from the nucleus of HEK 293T cells. Problem is whenever I am transfecting HEK 293T cells with overexpression plasmid, I am not able to enrich my protein from nuclear extract, probably because the protein is not going to the nucleus in sufficient amount after overexpression.
Kindly provide the inputs.
First, did you verify your fractionation by western blot for known nuclear and cytoplasmic markers? It is possible you lose your nuclear fraction due to problem in the protocol.
Second, do you know what is the mechanism by which your protein goes to the nucleus? If it's dependent on an importin that is lowly expressed than it may be rate limiting so there is not enough to take more of your overexpressed protein to the nucleus.
If this is the case you may need to either overexpress the importin or add a strong NLS to your construct.
Hi Yoav,
Answer to your first question is that the fractionation protocol which I am using is working very good for me. I have checked with cytoplasmic and nuclear markers on western blot. I am using the same protocol for isolating another protein which is solely nuclear. So yes the protocol is working good.
For your second question, the protein on which I am struggling does not have NLS. It goes to the nucleus by monoubiquitylation. As you said correctly, there can be a chance that the E3 ligase machinery is not enough for the overexpressed protein to send it to nucleus.
Attaching a strong NLS might work.
Thanks Yoav for your suggestion.
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