Cell Fractionation

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Erica
Erica's picture
Cell Fractionation

I'm studying a protein that transocates from the cytosol to the nucleus upon hormonal stimulation. I would like to know if this protein is associated with the cytoskeleton (particularly microtubules) in unstimulated cells --or-- if it associates with the cytoskeleton upon hormonal stimulation and is thereby transported to the nucleus.

My current protocol allows me to fractionate cells into triton-soluble and triton-insoluble fractions. The lysis buffer contains glycerol and other components that stabilize microtubules so that the microtubules are equally prevalent in both fractions. Treatment with colchicine causes depolymerization of the microtubules, resulting in their movement to the soluble fraction.

Are there any protocols or kits that allow for fractionation of the cell into distinct cytosol, nuclear, and cytoskeletal fractions AND will stabilize microtubules?

samm
samm's picture
Give a shot at Qiagens'

Give a shot at Qiagens' QProteome kits or the Pierce equivalent.
Do your antibodies work in IF? You might want to just stain your cells intracellularly with general tubulin Ab and one against your protein, under the different conditions. If it works, you could expand and quantitate by flow cytometry.

vanishing
vanishing's picture
I found this publication in

I found this publication in the medline, that has a protocol

Keiji Nishida * , Fumi Yagisawa * , Haruko Kuroiwa *, Toshiyuki Nagata , and Tsuneyoshi Kuroiwa *
Cell Cycle-regulated, Microtubule-independent Organelle Division in Cyanidioschyzon merolae

http://www.molbiolcell.org/cgi/content/full/16/5/2493