Ultrascan 2k 2k ccd camera

SpyCatcher-AP205 VLPs were expressed in bacteria and purified as described (48 (link), 88 (link)). SpyTagged SOSIP immunogens were incubated at a 2 to 3–fold molar excess with SpyCatcher-VLPs for 12 to 24 hours at room temperature in phosphate-buffered saline (PBS). Conjugated VLPs were separated from free Env trimers by SEC on a Superdex 200 (fig. S7D) or Superose 6 (fig. S7E) column. Conjugation of Env trimers was verified by SDS-PAGE (fig. S7C). Immunogen concentrations for immunizations were estimated by comparing to known amounts of the analogous unconjugated Env trimer on a SDS-PAGE gel.
Conjugated VLPs were examined by nsEM (fig. S7A and D). Purified VLPs were diluted to about 10 μg/mL immediately before adding 3 μL to a glow-discharged ultrathin C film on a holey carbon support film, 400 mesh, Cu grid (Ted Pella). After blotting, the grids were stained by uranyl acetate and then imaged using a FEI Tecnai T12 transmission electron microscope operating at 120 keV with a Gatan Ultrascan 2k × 2k CCD camera. Each image was collected using a 1 second exposure at about 2 μm defocus and 42,000× magnification, resulting in 2.5 Å per pixel.

GV samples were diluted to OD₅₀₀ ∼ 0.2 in 10 mM HEPES buffer and spotted on Formvar/Carbon 200 mesh grids (Ted Pella, Redding, CA), which were rendered hydrophilic by glow discharging (Emitek K100X). Unclustered Mega GVs were negatively stained using 2% uranyl acetate. Images were acquired using the Tecnai T12 LaB6 120 kV transmission electron microscope (TEM) equipped with a Gatan Ultrascan 2k × 2k CCD camera.