I am currently doing EMSA with pierce light shift EMSA kit. For binding reaction I use 20 pmol of biotin labeled DNA, 5 microgram of protein (his-tagged protein), hundred molar excess of unlabelled DNA , 50 ng of poly dIdC and the 1X binding buffer to the final reaction volume f 20 microlitre. I incubate it at room temperature for 30 min. After that I run it in 10% PAGE gel at 100 Volt ane then transferred to nylon memebrane with 0.5X TBE buffer and use that membrane for detection. First time when I did , I got good band shift in all the lanes which contain protein. Now in the repeatation experiment , I am getting a band shift in the control lane also which contains only the biotin labeled DNA WITHOUT any protein. I am attaching my blot here . What could be the possible reason for the band shift. Is it a non specific binding , or any protein contamination in my reagents. For the repeatatiopn experiment I used the same ).0.5X TBE buffer which I used for the first experiment. Please help.
Thanks in advance for your replies