Free DNA disappears but no band shift
 

Hi,

I have a question about EMSA. We are using recombinant protein (2 - 5 µg) 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 don´t 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 wasn´t visible after ethidium bromide staining.

Does anyone have a good idea?

Thanks in advance!

Re: Free DNA disappears but no band shift
 

hi,
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