I have been performing EMSA reactions with digoxigenin labeled DNA probe (240 bp) and His tagged purified protein (TyrR transcription factor) and have consistently been getting a weird extra band in my reactions. My labeled DNA always gives three discreet bands, of which only the smallest is shifted in the presence of TyrR. The additional bands do not seem to be the result of contaminating proteins binding, as the probes are extensively purified, nor are they the results of TyrR binding, which is conclusively demonstrated using mutated variants of the probe.
My supervisor and I are quite baffled by the presence of these bands, and am almost at my wits end trying to eliminate them from the reaction. Any suggestions of what they could be, or how to get rid of them would be greatly appreciated.
Attached is a EMSA image displaying the extraneous bands
Lane 1: DNA probe alone; Lane 2: probe + protein; Lane 3-6 control probe mutations + protein
As you can see, the free DNA probe (F) shows three distinct bands, the bottom of which declines in intensity when the protein is present and induces a shift.