In Article <7dfjnh$ons$1 at oyez.ccc.nottingham.ac.uk>, "Samuel Gray"
<samuel.gray at nottinghnospam.ac.uk> wrote:
>Does anyone have a good protocol for extracting total protein (for western
>blotting) from tissues frozen in liquid nitrogen? Should I pulverise the
>tissue whilst still frozen (as I would for RNA extraction) or allow the
>tissues to defrost and mince/sonicate in a protein extraction buffer?
I use a Polytron homogenizer with a small probe (which can disrupt a total
sample volume of 1 ml), and extract directly into boiling (heated in a
boiling waterbath that is) Laemmli sample buffer. Just put the frozen
tissue in a tube, add the appropriate volume of sample buffer, and
immediately homogenize. Then heat the sample in a boiling watrbath again
(because the tissue will have cooled the extraction down) and treat as I
would any other Laemmli sample. I think that the important element here is
to use a vigorous mechanical homogenization that will minimize the time that
the interior of the tissue thaws without having the hot SDS/mercaptoethanol
denaturing any proteases.
I've never had a problem with degraded bands using this approach, but on
the other hand I've never done an extensive study of alternatives either.
For what it's worth,
Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton, Alberta T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca
