Nuclear extracts/suspensions

[Youhua Xie]

Mathew Traini (mtraini at rna.bio.mq.edu.au) wrote:
: 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.
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.