Capture-stabilize approach for membrane protein SPR assays

Chu, Ruiyin; Reczek, David; Brondyk, William

In order to assess the impact of the avidity effect on this capture and stabilize approach, chip surfaces with two different receptor densities (300 and 700 RUs) were prepared. As shown in Figure 4a , at both receptor densities, high quality kinetics sensorgrams were generated with good reproducibility. The Kon-Koff rate map ( Figure 4b ) further highlighted the reproducibility of triplicate data points. Three antibody samples as well as the ligand were well-differentiated by this assay format, which was not possible using the two other peptide-based assay formats. The kinetics data shown in Table 3 further demonstrated the reliability of the assay regardless of the difference in receptor density. Therefore, this limited chemical crosslinking step generated a stable sensor chip surface with full ligand binding activity. Using this method, the stabilized chip surface was regenerated with 50 mM HCl for up to 2000 repeated cycles without any noticeable loss of binding activity.