Nucleus vs cytosol fractionation

Dear netters

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?

TIA

axexango wrote:


The fractionisation of cell compartments is discussed in several papers
in Meth. Enzymol. 31 (1974). However, in the end you will always have to
do assays for marker enzyme to confirm the purity of your fractions
(also described in that volume).

Dr Engelbert Buxbaum wrote:
for
papers
to

Thanks for the reference!

Just an update to my assays. I have almost no detectable RER
contamination in my nucleus fraction as determined by anti-calnexin
blots but I have cytosqueletton contamination as determined by
anti-actin. This latter contamination is problematic because I am
assaying for the nucleo-cytoplasmic shuttling of a cytosqueletton
associated protein.

I was thinking of using cytosqueletton destabilization drugs before
cell lysis in order to depolymerise it and prevent it's co-purification
with the nucleus fraction. Do you know if this might do the trick?
TIA

[Only registered users see links. ] wrote:

That's because there is plenty of actin in the nucleus. Gotta hate those
cytosqueletton proteins that aren't strictly cytosquelettal.

LOL

that's true! But I also have tubulin, is that part of the nucleus also?