Binding Affinity of Peptides to 14-3-3ε

The SPR analyses were carried out using the Biacore 8K surface plasmon resonance instrument (Cytiva, Marlborough, MA, USA). A total of 100 nM His₆-14-3-3ε was immobilized on the NiCl₂-activated NTA chip (Cytiva, Marlborough, MA, USA) as previously described [34 (link)]. Then, different concentrations of peptide in the range of 0−500 μM (depending on the peptide) were injected. The experiments were performed in 20 mM TRIS, 150 mM NaCl, 50 μM EDTA, 0.005% Tween 20, and pH 7.4 buffer. The affinities of the peptides binding to 14-3-3ε were determined by fitting the data to a steady-state binding model using Biacore Insight Evaluation Software (version 5.0.18.22102).