Ni nta nanogold beads

To visualize the effect of rAPOL3 addition to liposomes, liposomes containing 75:25 DMPC/DMPG (2 mM total lipid) were generated in 20 mM ammonium acetate and mixed with 40 μM rAPOL3 at 37°C for 5 min. The reaction was transferred to room temperature for an additional 30 min, then diluted 1/20 before loading onto glow-discharged copper coated EM grids (EMS, cat#CF400-Cu-50) and stained with 2% uranyl formate for 1 min. Grids were examined in JEOL1400 plus electron microscope with acceleration voltage of 80 kV. To visualize rAPOL3 on bacteria, log-phase E. coli^ΔhldE or Stm^ΔwaaL was incubated with 10 μM, 5 μM, or 2 μM rHis-APOL3 in 100 μl Buffer A for 5 min at room temperature. Bacteria were pelleted and blocked by resuspension in 360 μl 20 mM Tris pH 7.4, 10 mM imidazole, 200 mM NaCl containing 1.5% skim milk for 5 min at room temperature. 40 μl of 5 nm Ni-NTA nanogold beads (nanoprobes) was added for 10 min at room temperature and washed three times in 20 mM Tris pH 7.4, 20 mM imidazole, 200 mM NaCl before loading directly onto glow-discharged copper grids. Bacteria were treated with dialysate alone and processed in parallel to assess nonspecific binding of beads to bacteria.