Western - tiny tissue amounts

Henk Veldman H.Veldman at lab.azu.nl
Mon Feb 28 10:29:12 EST 2000


Jörg Kempfert schreef:

> First thanks for your interest.
>
> 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.
>
> Would be very kind if you could help us.
>
> Joerg Kempfert
>
> pathos schrieb:
>
> > In article <38B664E6.97A9B956 at uke.uni-hamburg.de>, =?iso-8859-1?Q?J=F6rg?=
> > Kempfert <kempfert at uke.uni-hamburg.de> wrote:
> >
> > > We want to do some western blot analyses with really small tissue
> > > amounts. Does anybody have experience with this kind of problem? It
> > > would be very nice if someone could help us in this case.
> > >
> > > Looking forward to get your hints.
> >
> > What is the problem?  I routinely use very small pieces of tissue for westerns.
> >
> > --
> > Peter Pediaditakis
> > University of Pittsburgh
> > Dept. of Pathology

I used to do SDS-PAGE - Western blotting on small (20-30mg) pieces of frozen muscle
biopsies. I took up the material in a 20 X volume of SDS-PAGE sample treatment buffer
(about 0.5ml) in a 4ml RIA tube. For homogenizing I used an Ultra-Turrax with a
dispersing tool for 0.3-3ml volumes at full speed  twice for 15 sec.
Hope this helps

Henk



--

H. Veldman
Laboratory for Experimental Neurology (NMZ)
Room G 02.320
University Medical Center Utrecht (AZU)
P.O. Box 85500
3508 GA  UTRECHT
The Netherlands
Tel: +31 30 2507565
Fax: +31 30 2542100
E-mail: H.Veldman at lab.azu.nl






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