Free DNA disappears but no band shift
I have a question about EMSA. We are using recombinant protein (2 - 5 ĩg) which might be a helicase and tested it for DNA-binding. As probe we used unlabelled PCR products of a lenght between 190 bp and approx. 300 bp (125 - 250 ng). We tried both agarose and native polyacrylamide gels in Laemmli running buffer without SDS.
With increasing protein concentrations we observed that the free DNA disappears but we donīt get a slower migrating band instead indicating protein-bound DNA. At high protein concentration there is no DNA at all.
When we stain the same gel with Coomassie afterwards we see a band corresponding to the protein that wasnīt visible after ethidium bromide staining.
Does anyone have a good idea?
Thanks in advance!
Re: Free DNA disappears but no band shift
i m having exactly the same problem...
The DNA start to disappear as i increase the amount of protein, without shifted bands
conditions: TrisHCl, Glycerol, NaCl, Tween20, DDT, poly dIdC,
so...do you have a solution for me ?
thanks in advance
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