Cell fractioning-Nuclei isolation

engelbert_buxbaum4 at my-deja.com engelbert_buxbaum4 at my-deja.com
Mon Nov 13 00:38:00 EST 2000


In article <3A0DCBD9.DDE1DA4C at med.unc.edu>,
  Stephen Snowdy <snowds at med.unc.edu> wrote:
> Hi folks.  I'm trying to isolate nuclei and cytoplasmic soluble and
> insoluble fractions in 293 cells by hypotonic swelling and Dounce
> homoginization. I'm concerned that I'm
> spinning down insoluble cytoplasmic stuff down with my nuclei.

There is a protocol for cell fractionisation in Meth. Enzymol. 31 (1974)
6-41 that should give you pure nuclei. Remember that the first
fractionated centrifugation will only enrich the pellet in certain
components, purification requires further steps (except for cytosol, of
course). However, do you actually need pure nuclei, i.e. would for
example plasma membrane contamination interfere with your assay?


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