Affinity Precipitation of SARS-CoV-2 RBD

Affinity precipitation assays using Ni-NTA beads (GE Healthcare) were performed with His-S RBD and FITC-SAPs using previously described methods.^{44 (link)} Ni-NTA beads were equilibrated in binding buffer (50 mM Tris pH 7.5, 250 mM NaCl, 50 mM imidazole, and 2 mM β-mercaptoethanol). Binding reactions were setup by incubating equilibrated Ni-NTA beads with His-S RBD (1 μM) and increasing concentrations (3, 1, 0.5, 0.25, and 0.125 mM) of indicated FITC-SAP in the binding buffer and incubated for 1 h at 4 °C. In parallel, control reactions with 3 mM of indicated FITC-SAP without His-S RBD were preformed to rule out nonspecific FITC-SAP binding to the Ni-NTA beads. Reactions were spun and washed three times in the binding buffer to remove unbound proteins/peptides. The resulting protein–peptide complexes bound to the beads were extracted using NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen), subjected to SDS-PAGE analysis, and visualized by Coomassie staining or fluorescence detection at 488 nm. Resulting FITC-labeled bands were quantified using ImageJ software. To estimate K_d values for FITC-SAP binding to His-S RBD, the data were fit to the Hill equation using Origin 8 software (OriginLab).