Cell fractioning-Nuclei isolation

Stephen Snowdy snowds at med.unc.edu
Sat Nov 11 17:44:41 EST 2000


Hi folks.  I'm trying to isolate nuclei and cytoplasmic soluble and
insoluble fractions in 293 cells by hypotonic swelling and Dounce
homoginization.  In Current Protocols, one protocol says to spin down
the nuclei at 3300 x g (nuclear extract assay) and another protocol in
the same book says to use 500 x g (run-off transcription assay).

When I spin at 3300 x g, the pellet has two layers:  an opaque white one
at the bottom, and a yellower one on top.  I'm concerned that I'm
spinning down insoluble cytoplasmic stuff down with my nuclei.

Is anyone familiar enough with isolating nuclei to help me out?

Thank you for taking the time.
Steve






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