Nuclear extracts/suspensions

[Mathew Traini]

Hi...

I'm trying to prepare a nuclear extract from CHO cells, but 
I'm not sure if the protocol I'm currently using is actually
working.

Briefly, the cells are spun down, washed with saline, and then
resuspended in 0.3% NP40 detergent, Tris, MgCl2, NaCl, DTT, and 
a cocktail of protease inhibitors.  This suspension  is then
pushed rapidly through a small gauge needle 5-6 times, which
I am told will shear the cell membrane, leaving nuclei intact.

The nuclei are then spun down, washed, and resuspended in a 
40% glycerol buffer for storage at -70C.  I did a Lowry assay
on the fractions...the cytoplasmic fraction was extremely rich 
in protein (as would be expected), but the nuclear suspensions 
(which had been thawed after being frozen at -70C) showed 
almost baseline levels of protein.

Does anyone think this method will actually separate out 
nuclei?  I got it from a manual, and it looked quite easy, so I 
gave it a go, but the protein assay results might suggest that 
in fact everything ended up in the cytoplasmic fraction, and 
the small pellet which I thought should be nuclei was in fact 
just membranes and other crap.

Any responses or suggestions appreciated.

Mat,
School of Biological Sciences,
Macquarie University,
NSW, 2109.