xie at assel.biochem.mpg.de
Wed Nov 6 05:40:39 EST 1996
Mathew Traini (mtraini at rna.bio.mq.edu.au) wrote:
: 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.
You may try a dounce in stead of needles to destroy the cell membrane.
Check if the nuclei are intact under microscope the first time you
use a dounce and determine how many strokes you need. I never use
any detergent. It works fine for my HepG2 cells.
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