BR-1 chemical bead preparation and protein binding detection

BR-1 and control beads were prepared as described^31,32. Briefly, 100 μl of photoaffinity linker-coated agarose beads was suspended in 200 μl of isopropyl alcohol and dried in vacuo. Methanol (control beads) or BR-1 solution (1 mg/150 μl methanol) was added, and the mixture was dried in vacuo. The beads were irradiated at 365 nm using an ultraviolet (UV)-activated crosslinker (4 J/cm²). The irradiated beads were sequentially washed in 50% methanol, methanol, DMSO, and methanol (3 × 400 μl/wash) and suspended in 200 μl PBS (1.8 mM KH₂PO₄, 10 mM Na₂HPO₄•12H₂O, 137 mM NaCl, and 2.7 mM KCl). To examine protein binding, 25 µl control beads or BR-1-conjugated beads was mixed with cell lysates (2 mg protein) from S. lividans TK23 cells expressing the RevU protein and incubated overnight at 4 °C in 1 ml binding buffer. Then, the beads were washed 5 times with binding buffer containing 0.1% Tween-20. The bound proteins were eluted with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The eluted protein was analysed by 7.5% SDS-PAGE. To identify proteins, MALDI-TOF/MS analysis was performed using an Ultraflex instrument (Bruker Daltonics).