Label-free Binding Assay for Prion Protein

The detection of binding events at the equilibrium was performed using the label free, DMR module of an EnSight Multimode Plate Reader (Perkin Elmer, MA, USA) [25 (link)]. Mouse or human recombinant PrP were immobilized onto the surface of DMR plates (15 μL/well of a 2.5 μM recombinant PrP solution in 10 mM sodium acetate buffer, pH 5) using an amine-coupling chemistry. The interaction between CPZ and recombinant PrP or BSA was evaluated by incubating different concentrations of the compound (0.1–2,000 μM; diluted in assay buffer: 10 mM PO4, pH 7.5, 2.4 mM KCl, 138 mM NaCl, 0.05% Tween-20) for 30 min at room temperature. All steps were performed by using a Zephyr Compact Liquid Handling Workstation (Perkin Elmer). Final signals were obtained by automatic intra-well, empty surface normalization, and by subtraction of the control wells (no protein immobilized, or vehicle added). The Kaleido software (Perkin Elmer) was used to acquire and process the data.