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[jim_reaper1066]

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.

Attached Files

•  emsa2.001.tiff   543.83K   108 downloads

[pcrman]

I am little confused. Why do you say that one of the smallest band in the free DNA area is the shifted band? Free probes are just unbound probe and won't exhibit shift but only change in amount. What you expect to see are bands above the free probe area. The extra bands in the free probe area could represent different forms of the probe: single or double-stranded.