Hi all,
I am currently investigating a protein that shows both nucleolar and nucleoplasmic staining, and one of the analyses I would like to conduct is to test whether the nucleoplasmic fraction is chromatin-associated or free in the nuclear milieu.
My first plan is to conduct a Chromatin Fractionation Assay as described by Mendez and Stillman (2000). However, I have not been able to find any information, either on forums or in papers, about how nucleolar proteins (and thus, how my nucleolar protein fraction) behave in this experiment.
Has anyone performed this analysis on nucleolar proteins and established where they segregate? Does the nucleolus remain intact and precipitate with the (insoluble) chromatin? Does it remain in the soluble fraction? Does it dissociate and appear in both nucleoplasmic and chromatin fractions?
Anyone able to share their experience in this area would be greatly appreciated!
Alternatively, other suggestions on how to better investigate this question are also welcome - I'm alternatively considering Chromatin Retention Assays (Andegeko et al. 2001) or Histone Association Assays (Ricke and Bielinsky, 2005).
Many thanks in advance!
Dan
I‘ ve conducted chromatin fractionation to investigate whether a protein localizes on the chromatin during mitosis. GAPDH can be used as the cytoplasm marker and H3 as the chromatin marker in western bloting detection. Not only that, I think you should also use some known nucleolar protein as the nucleolus marker, then detect which marker the protein you are interested stay with.
AND.............
Why don't you conduct CHIP for this protein and detect whether it interacts with rDNA ?But, I don't know whether CHIP can help you.
Hi Cai,
Thanks for your message! Just to clarify, I'm trying to see if the nucleoplasmic staining we see is chromatin associated or free in the nucleoplasm, rather than if the nucleolar staining is rDNA associated; however to do that, I need to know where the nucleolar staining segregates with the nucleoplasmic or chromatin fractions (and thus whether it would mask the presence of the nucleoplasmic fraction). I'll definitely try it with a nucleolar marker, as you sugges, but just want to see if anyone else has tried it previously and could provide some guidance.
Running a ChIP is something that I'm certainly considering; however, Id like to have clearer evidence of definite chromatin association before I dive into that area.
all the best,
Dan
Hi All,
Just to update - I performed a modified Mendez and Stillman method and found that most nucleolar proteins (at least, those in the DFC/GC - e.g. NPM1, NOL12) segregated predominantly to the nucleoplasmic fraction rather than the chromatin one.
Cheers,
Dan
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