help needed on gel retardation assay

gc genecutl at
Fri Mar 25 11:54:12 EST 1994

In article <2mvop3INNcgp at dns1.NMSU.Edu>, twei at kirk (Tao Wei) wrote:

> Dear netters:
> I am a graduate student and working with tomato plant.  I am running into  
> a problem in gel retardation experiments.  The DNA probe (200 bp prepared  
> by PCR) and crude nuclear proteins seemed to form very large complexes  
> (because of non-specific interaction?), stuck on the bottom of wells and  
> could not get into the gel.  The condition of binding reaction was pH 7.5,  
> 50 mM NaCl, 9-11% glycerol and some competitor DNA.  Does anybody have any  
> suggestions to help me out? Thank in advance.

Try using NP-40 in your binding buffer and running buffer.  Have you
tried titrating the protein levels?  Are there no concentrations 
between nothing bound and everything bound in the wells?  WIth any
DNA-binding protein, if you add enough of it, it'll shift everything
to the top of the wells.  You can tell if this is happening if there
isn't any free probe at the bottom.  For best results, you don't
want more than half your probe shifted.

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