Ni-NTA Agarose (15 uL buffer equilibrated bead slurry) was incubated with 3 μM RAD52, 3 μM RPA in the presence or absence of 3 μM ‘1’ or ‘6’, in the binding buffer (30 mM Tris-Acetate pH7.5, 1 mM βME, 150 mM KCl, 30 mM Imidazole, 5% glycerol, and 0.2% Nonidet P40 substitute). After 30 min incubation on a neutator at 4°C samples were spun down, and the aliquots of unbound ('free') proteins from each reaction were saved. Then the beads were washed and the bound proteins were eluted with 20 uL elution buffer (the same as the binding buffer, but with 400 mM Imidazole) and saved for gel electrophoresis. Free proteins and proteins co-eluted from the beads ('bound') were separated on the 12% SDS PAGE gel. Lane 1 is a loading control, which shows RAD52 and the three subunits of RPA (RPA70, RPA35, and RPA14). The proteins and the compounds present in each reaction are indicated in the table above the gel. The carton on the left of the gel schematically depicts the experiment: RAD52 protein binds to the Ni-NTA beads through the interaction with its 6xHis tag; RPA is untagged and can be retained on the beads only through a specific interaction with RAD52 (Grimme et al., 2010). The experiment was repeated three times (a representative gel is shown) and no change in the ratio of RAD52 and RPA co-eluted from the beads in the presence and absence of ‘1’ or ‘6’ was detected.