Ni nta nanogold particles

Images were obtained with a Technai G2 Spirit instrument operating at 80 kV. Samples were freshly diluted in Bicine buffer. Drops (10 μL) of the sample solution in Bicine buffer were applied to carbon coated copper grids (Ted Pella, Inc.) for 2 min before removing the excess solution with filter paper. Images of Ni–NTA Nanogold^® particles (Nanoprobes, Inc.) bound to RubisCO or PRK, which were subsequently associated with the nanostructures, were obtained without the need for prior staining. Nanogold particles were pre-bound to histidine-tagged proteins by following the procedures described elsewhere (Nanoprobes, Inc.). Ni–NTA Nanogold^® particles mixed with just the nanostructures in the absence of histidine-tagged proteins provided evidence for the absence of non-specific association. For samples prepared without Nanogold particles, uranyl acetate was used to stain the negatively-charged groups. To accomplish this, the grid containing the sample was floated on 10 μL drops of a 2% (w/v) uranyl acetate solution for 1 min before being imaged by TEM. Several replicates were imaged from at least 2 independent preparations to ensure that the imaging pattern was consistent for each sample.

Purified C-His₆-CydDC was mixed with 5 nm Ni-NTA Nanogold particles (Nanoprobes, Yaphank, NY) at a molar ratio of 2:1 (final concentrations of 56 and 28 μm, respectively) in 20 mm Tris-HCl (pH 7.6), 20% glycerol, 150 mm NaCl, and 0.02% DDM and incubated briefly at room temperature. The mixture was concentrated in a Viva Spin 4 concentrator (MWCO 100,000) to 0.5 ml and clarified by centrifugation at 16,000 × g for 10 min at 4 °C. Unbound gold particles were separated by applying the supernatant onto a Superose 12 10/300 GL column (GE Healthcare) equilibrated with 20 mm Tris-HCl (pH 7.6), 20% glycerol, 300 mm NaCl, 10 mm imidazole, and 0.02% DDM. Separation was performed on an ÄKTA FPLC system at a flow rate of 0.3 ml/min. Labeling of the C-His₆-CydDC-EPL proteoliposomes was performed as above, but the glycerol and DDM were omitted from the binding buffer, and the free gold label was removed by washing the liposomes three times in 20 mm Tris-HCl (pH 7.6), 300 mm NaCl, and 10 mm imidazole by passing the pellet through a 27-gauge needle several times, followed by centrifugation at 16,000 × g for 10 min at 4 °C.