Southwesterns

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Thu Apr 10 11:24:12 EST 1997


In article <5idmbg$1ltm at news.doit.wisc.edu>, hakorsmo at facstaff.wisc.edu
(Helen) wrote:

> We perform Southwesterns on rat intestinal nuclear extracts and get a
band at about 30 kDa 
> (which we believe to be the correct size for the DNA-binding protein we
are working with), but 
> also  a very intense band showing up on the autorad at a molecular size 
of about 15 kDa.  
> Could this band be due to non-specific binding of our  P32-labeled oligo
to Histones? I have 
> seen similar bands in some articles, but this is not always the case.  
Is there any way to get rid 
> of this band? Are there any good references for Southwesterns that you
can recommend?
> Thanks,
> Helen

Hi Helen,
We´ve done lots of Southwesterns in the last years. The quality of these
blots is highly dependent on your conditions and the DNA you use.
A buffer that works great in our hands is simply (10mM Tris-Cl (pH 8.0),
80mM NaCl, 2mM EDTA). Be sure to use sufficient, but not to much labelled
probe (we use 200.000cpm per membrane of 8x10cm). Use competitor DNA if
you want to see specific protein bands (yes, histone may shine up with
some probes, although unspecifically). As a starting point I would suggest
a 1000fold mass excess of sheared E.coli DNA. 

Hope this helps,
Frank

-- 
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
D-78434 Konstanz
Germany



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