i am doing the EMSA assay which made me mad, i used the Cy5 labeled primers to amplify the fragment for probe,PCR product was run on the agarose to confirm the unique PCR product. 1ul PCR product(with out gel purification) was used for EMSA, i always got additional band above the free probe, when i added less 0.5 ul PCR for EMSA, this additional band was less as well, do how can i address this problem? is it nessary to purify the PCR product before EMSA?
PS: lane1 and 4: only probe without protein
lande2 and 5: 0.025ug protein + 1ul and 0.5ul probe respectively
lane3 and 6 : 1ug protein + 1ul and 0.5ul probe respectively
waiting for your nice suggestions!