Streptavidin rpe

On day 4, yeast-HA complexes were pelleted at 4°C and washed twice with 5% PBSA + 2 mM EDTA. Complexes were then incubated with Streptavidin-RPE (1:100, Thermo Fisher Scientific, Waltham, MA, #S866) and anti-cMyc-FITC (1:50, Miltenyi Biotec, Somerville, MA, #130-116-485) at 4°C for 45 min in the dark. Following incubation, complexes were washed twice with 5% PBSA + 2 mM EDTA and stored on ice in the dark until sorting.

After overnight induction, yeast cultures were pelleted, washed twice with 0.01% PBSA (VWR #45001–130; GoldBio, St. Louis, MO, #A-420–50), and resuspended to an OD600 of 1. A total of 500-700 μL of OD1 yeast cells were labeled with biotinylated human ACE2 (Acrobiosystems #AC2-H2H82E6) at each of the twelve ACE2 concentrations (half-log increments spanning 10^−12.5–10⁻⁷M), with volumes adjusted to limit ligand depletion effects to be less than 10% (assuming 50,000 surface RBD/cell^{37 (link)}). Yeast-ACE2 mixtures were incubated and rotated at room temperature for 20 hr. Following the incubation, yeast-ACE2 complexes were pelleted by spinning at 3000 × g for 10 min at 4 °C, washed twice with 0.5% PBSA + 2 mM EDTA, and subsequently labeled with Streptavidin-RPE (1:100, Thermo Fisher #S866) and anti-cMyc-FITC (1:50, Miltenyi Biotec, Somerville, MA, #130-116-485) at 4 °C for 45 min. After this secondary labeling, yeast were washed twice with 0.5% PBSA + 2 mM EDTA and left on ice in the dark until sorting.

On day 4, yeast-HA complexes were pelleted by spinning at 3000 x g for 10 min at 4°C, washed twice with 5% PBSA + 2 mM EDTA, and simultaneously labeled with Streptavidin-RPE (1:100, Thermo Fisher #S866) and anti-cMyc-FITC (1:50, Miltenyi Biotec, Somerville, MA, #130-116-485) at 4°C for 45 min. Following secondary labeling, yeast were washed twice with 5% PBSA + 2 mM EDTA, and left on ice in the dark until sorting.