Western - tiny tissue amounts

[pathos]

In article <38BA53DA.4711722A at uke.uni-hamburg.de>, =?iso-8859-1?Q?J=F6rg?=
Kempfert <kempfert at uke.uni-hamburg.de> wrote:

> The main problem for us is: how to crush those small pieces of tissue
(here talking
> about 20mg each) and then to get the cytosol.

I use 2ml Homogination buffer for 0.2g of tissue.  If I were you, I would
use 0.4 ml of homgenization buffer and use a polytron microtip. The
homogenzation bufffer is 20 mM tris pH 8 with protease inhibitors.  If you
decide to use detergents, then you will get membrane proteins as well. 
Without detergent, you would get largely cytosolic proteins.
After a breif homgenization(20-30 sec), spin for about 30min at at least
15000g.  Discard the pellet and the suopernatant should be "mostly"
cytosol.
If you do not have a concentrated enough supernatant, then you can reduce
the volume by dialysis or phenol ether extraction.

If you need more, please ask.


Regards,

-- 
Peter Pediaditakis
University of Pittsburgh
Dept. of Pathology