Nucleus vs cytosol fractionation

axexango at hotmail.com axexango at hotmail.com
Tue Feb 1 19:57:52 EST 2005


Dr Engelbert Buxbaum wrote:
> axexango wrote:
>
> > 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?
>
> 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).

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




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