What is the charge on your protein? If it is a very positively charged protein it may not enter the gel. I have found this before in EMSAs but normally at low protein concentrations it still enters the gel only at the higher concs it does not. I had to change protein to DNA ratio and also the percentage of the gel. There was quite a bit of optimization that had to be done.
Sent from my iPhone > On 13 Feb 2015, at 11:50 PM, "Sudipta Bhattacharyya" > <[email protected]> wrote: > > Dear Community, > > Sorry for this off topic question. I am dealing with a non specific DNA > binding protein. In a non radio-labelled EMSA DNA:protein titration > experiment when I EtBr stained the 5% native acrylamide gel, I could see the > DNA control (101 bps) but as the amount of my protein increases I saw the > free DNA band getting thinner and thinner but I could not see any band shift > and probably all the complex stuck at wells. But, even if I assume the whole > DNA length is occupied by my protein (I determined the ratio to be 1:15 ish) > the molecular weight should not reach the molecular weight of the highest DNA > marker band (20kbps = 13,200 kDa). Then why I could not see a retarded band > in the gel? Or is it forming a EtBr resistant nucleoprotein filament? Could > you guys please give me an idea/suggestions? > > Many thanks in advance...!!! > > My best regards, > Sudipta. <table width="100%" border="0" cellspacing="0" cellpadding="0" style="width:100%;"> <tr> <td align="left" style="text-align:justify;"><font face="arial,sans-serif" size="1" color="#999999"><span style="font-size:11px;">This communication is intended for the addressee only. It is confidential. If you have received this communication in error, please notify us immediately and destroy the original message. You may not copy or disseminate this communication without the permission of the University. Only authorised signatories are competent to enter into agreements on behalf of the University and recipients are thus advised that the content of this message may not be legally binding on the University and may contain the personal views and opinions of the author, which are not necessarily the views and opinions of The University of the Witwatersrand, Johannesburg. All agreements between the University and outsiders are subject to South African Law unless the University agrees in writing to the contrary. </span></font></td> </tr> </table
