Can I extract nuclear proteins in transfected cells? - (Mar/28/2006 )
My study focuses on the effects of LANA inactivation of GSK-3beta kinase activity on the subcellular localization of beta-catenin.
I want to transfect a cell line (say 293T cells) with LANA-expression plasmid to study the changes of subcellular localization of beta-catenin. For this reason, I will need to extract both cytoplasmic and nuclear proteins for western blotting.
I am wondering if I can do cytoplasm and nuclear extraction in transfected cells?
a lysis buffer containing a detergent such as nonidet NP40 will permeabilise cell membranes but not nuclear membranes - then the nuclei can be spun out and protein extracted with an SDS buffer
eg. 10mM tric-cl pH7.4, 3mM CaChloride, 2mM Mgchloride, 0.5% Nonidet NP40
or you can use a lysis buffer such as Passive lysis buffer, if you have any Promega luciferase assay kits hanging about the lab...
i posted my protocols, and apply them on transfected, native or infected cells.