EMSA Binding Analysis of Stl Oligonucleotide

EMSA experiments were done using an 183mer oligonucleotide (Stl binding site₁₈₃) derived from the 171mer oligonucleotide described previously (5 (link)). Stl binding site₁₈₃ (75 ng) and the investigated proteins were mixed in EMSA buffer (PBS (pH 7.3), 5 mM MgCl₂, 75 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid) in the presence or absence of α,β-imido-dUTP (dUPNPP) in 20 μl total volume. Before loading onto 8% polyacrylamide gel the samples were incubated for 15 min at room temperature. Electrophoresis was performed in Tris- Borate- EDTA (TBE) buffer for about 60 min at room temperature, after 1 h pre-electrophoresis. Gels were detected with a Uvi-Tec gel-documentation system (Cleaver Scientific Ltd., Rugby, UK) using GelRed staining (Biotium).