Does someone know how to compare the amount of a protein in the nuclei of different cell lines with immunofluorescence?
Is it all right to perform the comparison basing on the ratio of nucleus:cytoplasm from each cell line? What are the points I need to pay special attention to?
To some degree this is okay. It is commonly done for looking at nuclear translocation of NFkB for example. You need to be certain that you are getting an honest representation of the distribution state by collecting z-stacks through the entire cell, however. Nuclei can be positioned very heterogenously in even a single field and if you only take a single image, you may skew your data.
Another option is to do cell fractionation and look for the quantity of your protein in the nuclear fraction versus lamin by western blotting.
Dr. Jordan R. Yaron, thank you a lot for the suggestions.
I just collected the z-stacks (10 stacks) around the center of the nuclei but not the entire cell, is it possible for me to find the representative distribution among them?
Also, I have planned to do the western blotting to quantify the target protein from the cell fractions.
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