Western - tiny tissue amounts
pathos at freerealtime.com
Mon Feb 28 10:35:20 EST 2000
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
> 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"
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.
University of Pittsburgh
Dept. of Pathology
More information about the Methods