I am using LightShift Chemiluminescent EMSA (Thermo Scientific, USA) kit for showing DNA-protein interaction of a transcription factor to promoter of a gene. A double stranded 30 bp oligonucleotide probe was prepared by biotinylation of single stranded oligonucleotides following by annealing (Pierce's Biotin 3' End DNA Labeling Kit). EMSA was performed by binding the probe with a recombinant protein and running samples in 5% gel using 0.5X TBE buffer (45 mM Tris, pH 7.5, 45 mM boric acid, 2 mM EDTA). Briefly, each reaction contained 2 μl of 10X binding buffer, 1.0 μl each of 1 µg/µl poly(dI-dC), 50%glycerol, 1%NP-40, 1M KCl, 100 mM MgCl2, 200 mM EDTA, 3 µl (3 μg) purified protein, and 2 µl (20 fmol) labeled probe or unlabeled DNA as the competitor made up to a final volume of 20 µl. The binding reactions were incubated at 25°C for 20 min.

I've run my EMSAs alongside EBNA controls (which are included in the LightShift EMSA kit).

After transfer to positively charged nitrocellulose membrane and developing, it seems that my samples are stuck close to wells. I repeated it 2-3 times (increased the gel running time also) but same results again and again.

Can someone help me troubleshoot this problem. I'll appreciate if you can provide detailed protocol if you have any. 

Thanks in advance