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-   -   Free DNA disappears but no band shift (http://www.molecularstation.com/forum/electrophoretic-mobility-shift-assay-forum/45026-free-dna-disappears-but-no-band-shift.html)

ATV 11-14-2008 04:17 PM

Free DNA disappears but no band shift
 
Hi,

I have a question about EMSA. We are using recombinant protein (2 - 5 g) 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 dont 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 wasnt visible after ethidium bromide staining.

Does anyone have a good idea?

Thanks in advance!

choco 12-14-2009 09:58 PM

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


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