Transcriptionally active nuclei from Adipose tissue

Dr. D.G. Fernig dgfernig at liverpool.ac.uk
Tue Dec 20 13:08:25 EST 1994


hAPPy gUY (ftajb at aurora.alaska.edu) wrote:
:  I am running into difficulties using sucrose gradients, but
: only with adipose tissue. Typically, the
: nuclei from adipose move upward in the gradient with the lipid layer and I
: have yet to come up with a trick to make them do otherwise.

The first rule is don't use primary cultures of adipocytes if you
can avoid it :-).  If you have to, the first thing is to defat the
homogenate before you go near a sucrose gradient.  Easiest way: 
homogenise adipocytes (a few strokes at 4C will do), then centrifuge
5 min ca 1000-3000 rpm.  at 4C.  You should have a large, solid fat
cake at the top of your tube.  Now either collect the infranatant and
the pellet, OR carefully remove the fat cake and drippings of fat
around the top of the tube (I prefer this way) and transfer the ioonfra-
natant and pellet to another tube.
Dave



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