How can I separate nuclei from cytoplasm in trophoblastic cells - (May/08/2007 )
I'm a new student in this field and I'm working on Beta-catenin expression in trophobastic cells. This protein has both cytosolic-membranous and nuclear expression. To study this protein, first I need to isolate nuclei from cytoplasm the way that I could keep both part and not to have contamination from each and then load them for Western . I searched on internet but I got confused. If anybody can guide me about nuclear isolation and extraction, I would appreciate.
Hi nemesis52 and everyone,
I'm also working with beta-catenin in rat liver lysates. Since this protein has cytosolic-membranous and nuclear expression I'd performed subcellular fractionation in order to obtain 1) plasma membrane (PM), 2) cytosolic fraction and 3) nuclear extract.
By western blotting I'd found a strong band corresponding to beta-catenin in PM fraction, a weaker band in cytosol fraction and, contrary to all reports, a strong band in nuclear extracts. The rats had no treatment at all and they are all adults. I had performed some LDH activity assay to make sure my nuclear extracts are not contaminated with cytosol fraction. I also check the purity of my nuclear extracts making an immunoblotting against a nuclaer transcription factor, and they had no contamination.
I'm wondering if this band could be arise from a PM contamination. In this case, what should I do? Can I use gamma-GT as a PM marker by WB?
Does anyone have and want to share a good protocol to obtain nuclear extracts better than mine?
Thanks in advance!