EMSA

Electrophoretic mobility shift assay (EMSA) assays were essentially performed as described before (22). In short, 120 ng of the polymerase chain reaction fragment was incubated for 20 min at RT in Binding Buffer (20 mM Tris HCl [pH 8.0], 1 mM EDTA, 5 mM MgCl₂, 100 mM KCl, 10% (v/v) glycerol) without and with increasing amounts of purified His₍₆₎Reg₅₇₆ in a total volume of 20 μl. Next, samples were incubated on ice for 10 min and were subsequently loaded onto a 2% agarose gel in 0.5·TBE. Electrophoresis was carried out in 0.5·TBE at 60 V at 4°C. After electrophoresis, the gel was stained with ethidium bromide, de-stained in 0.5·TBE and photographed under UV illumination.