I am attempting to fractionate the nucleus from the cytosol in order
to reveal a shift in subcellular localisation of my protein of
interest after RNAi of a given protein partner.
I know this RNAi induces a shift when I look at transfected fusion
protein under the microscope. But I want to make sure it really does
through a biochemical assay. The problem is that my protein migth be
associated with ER through RNA-independent ribosome binding.
I have some doubts as to the protocol to use to lyse the cells. I am
using mouse N2A cells. I thought about using the protocol in Robb et
al (2005, Nat Stuct & Mol Biol, january) as the PI, in an interview
given to the scientist, argues that the protocol followed for that
study is based on published and controlled protocols for fractionation
of ER from nucleus and is even harsher. But since they do not show
controls for ER contamination in their nuclear fraction, I'm not sure
as to whether I should believe in it.
Do any of you know of published protocols that has been controlled for
ER contamination in the nuclear fraction and allow to have highly
enriched cytosol vs nuclear fractions?