mtraini at rna.bio.mq.edu.au
Tue Nov 5 09:54:09 EST 1996
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
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.
School of Biological Sciences,
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