Huge Smear in EMSA - (Nov/25/2013 )
I am using biotin labeled oligos and I am getting just a giant smear all the way down the lane. I have tried diluting the amount of biotin labeled oligo I'm using and while the intensity of the smear decreases, there is still a smear down the entire lane. I can tell that there are bands at certain places but I would like to clean it up. I am using 2ug protein and poly di/dc. Would it be helpful to add in other components to the binding reaction or should I continue to try diluting my biotin labeled oligos?
Any help is greatly appreciated!!
How about the lane with oligo minus proteins - do you get similar smear? Are you using nuclear extracts or other protein?
I have a similar problem (please see attached image). I am not using labelled oligos and am simply staining the gel with ethidium bromide. The protein is affinity purified and I've run an SDS-PAGE to make sure there's no protein degradation. Why is the noDNA/protein only lane lighting up?
Any insights will be greatly appreciated