I am over-expressing a protein of interest (implicated in DNA repair) in fusion with yfp in Nicotiana Benthamiana leaves. The proteomics data showed that the protein is cytoplasmic as well as chromatin assotiated. The microscopic studies done at the same time showed high cytoplasmic expression for the protein but couldn't detect the protein in the nucleus. Under YFP filter, I can see fluorescence around the nucleus and faint color inside. I used as a control for nuclear localisation mcherry "expressed by the same construct" which showed very bright fluorescence. I am just wondering if there is a technical issue that hinder the bright nuclear fluorescene (could be the high cytoplasmic fluorescence!) and how can I optimize the experiment to be able to see the nuclear fluorescence. Thank you.
It probably is tough to see the nuclear signal next to all the cytoplasmid "background". Do you know the chromatin binding domain/nuclear localisation signal domain/sequence on your protein? I would test just that domain as a fusion protein with YFP and see if only the nucleus lights up..
I guess in this case it may be hard to solve the problem just by technical tweaking since the high cytoplasm/nucleus ratio seems to be the property of protein, i.e. only small proportion of the protein is localized in nucleus. Maybe different approaches could be tried to study the protein specifically in the nucleus, for example by adding strong nuclear loaclization signal (from any nuclear protein) in the construct.
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