Yes it can. Ethidium stains DNA by intercalating between base pairs, so if most of your DNA is tightly bound by protein the ethidium may not be able to wiggle in there. We've seen that happen with Mu transposase - DNA complexes. If that's the problem, soak your gel in SDS plus EtBr and you should see the DNA.
Unfortunately, if your protein and DNA are aggregating in the wells, there is a possibility of the staining process knocking them loose. In that case, you'll need to play with the conditions to get nice, soluble complexes that enter the gel (tweak the [salt], try adding a little non-denaturing detergent, change the pH, speak to it kindly but forcefully ...). Some DNA-binding proteins are not soluble at high concentration and low salt, so you may need to lower the concentration of everything in your assay, which may require switching to a more sensitive stain (or 32P). Also, because the mobility in a native gel is determined by the charge: mass ratio and the shape, it is impossible to predict exactly where the complex should run (i.e. your DNA-only markers are meaningless except for marking the free-DNA band). Good luck! ++++++++++++++++++++++++++++++++++++++++++ Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago pr...@uchicago.edu<mailto:pr...@uchicago.edu> ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sudipta Bhattacharyya [sudiptabhattacharyya.iit...@gmail.com] Sent: Friday, February 13, 2015 3:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Can nucleoprotein complex inhibit EtBr binding? 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.
