Cell fractioning-Nuclei isolation

engelbert_buxbaum4 at my-deja.com engelbert_buxbaum4 at my-deja.com
Tue Nov 14 00:13:50 EST 2000


In article <3A10AAEB.D6539A2E at med.unc.edu>,
  Stephen Snowdy <snowds at med.unc.edu> wrote:
> I'm actually just trying to find out which compartement a protein
> trafficks to after coming off the ribosome (w/out using IHC), i.e.
does
> it go to the nucleus, or does it end up in a cytoplasmic soluble or
> insoluble fraction.  So I guess I just need to make sure that no
> cytoplasmic soluble or insoluble components are in with my nuclei.  I
> think the way I'm doing it (spinning nuclei down at 3300 x g) ends up
> pelleting the mitochondria down with the nuclei, but I'm not really
> sure.  Any help?


Both mitochondria and plasma membrane are easily spun down with nuclei
in the initial low speed spin. If you are just intrested to see whether
your protein is cytosolic or enters one of these organelles, that does
not need to worry you, just wash the pellet 2 or 3 times to remove all
soluble material and you are fine. Similarly, you may wish to check the
high speed supernatant (100,000 g 15 min, for example in a
mini-ultracentrifuge) for the presence of your protein.

Once you found your protein in the pellet, you may want to identify the
orgnelle it goes to, and then that Fleischer & Kervina paper I quoted
should come handy, as it allows you to isolate pure organelles. In this
case you may want to carefully take note of the enrichment of marker
enzymes during the purificaton, and compare it with the enrichment of
your protein.


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