Hello All, I need help with isolating nucleoli from cancer cell line, I want a pure fraction devoid of nuclear or cytoplasmic contamination. I tried several times with this protocol " Li ZF, Lam YW. A new rapid method for isolating nucleoli. Methods Mol Biol. 2015;1228:35-42. doi: 10.1007/978-1-4939-1680-1_4. PMID: 25311120. " However not able to get a pure fraction, how it is shown in the protocol. Every time I am getting contaminant from nucleus and cytoplasm. I am using FBL as marker of nucleolus, LAMIN B, and FUS/TLS for nucleus and actin and GAPDH as marker of cytoplasm. Although enrichment in FBL is very prominent. But I need a clear nucleolar fraction.
Any suggestion will be appreciated.