Question about gel shift assay

[Lynne_M_Roxbee_Cox]

SAI-Jinling Xie wrote:
> 
> Hi, there,
> 
>      We've been doing gel shift assay. But we always got non-specific
> shifted band. We used more than 220bp PCR products as probes and crude
> cell extract from E.coli for shifting. A company recommends very short
> probes. I wonder if our probes are not short enough or some reasons else.
> And does impurity of the protein cause this problem? Do I have to purify the
> protein? If somebody happens to have the similar experience, would you
> please reply to this account? I would highly appreicite it. Thanks a lot
> in advance.
> 
> Jinling Xie

Jinling,

I did gel shifts with oligos that were about 30-40bp but I don't think that the DNA 
is your probem.

Lots of proteins bind to DNA - any DNA that happens to be around and you're likely 
to have lots of these in an E.coli extract.  You may not need to purify the 
protein, just add some "non-specific DNA" to your reaction tubes - for example 
herring testes DNA or calf thymus DNA.  That's want I did for mine and it worked a 
treat.  

I did still get a couple of non-specific bands - these could be shown to be 
non-specific by adding un-labelled probe to the reaction in excess (about 200 
fold).  This will cause competition and the specific bands will dissappear, the 
non-specific ones won't.

I hope this is of some help to you.

Lynne
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