Binding of human P-selectin to immobilized TM11 peptide and rPSGL-Ig.
Biotinylated TM11 was bound to the sensor surface through conjugation to immobilized streptavidin (A,C,E). rPSGL-Ig was directly immobilized onto the sensor chip (B,D,F). Soluble P-selectin (sPS) and PS-IgG (PS) were injected over both surfaces at a flow rate of 20 μL/min for 30 and 150 seconds, respectively, as indicated by the horizontal bar. Bound selectins were removed by a short injection of 10 mM NaOH (not shown) and were reused for subsequent injections. The figure shows an overlay of the sensorgrams. (A,B) Increasing concentrations of sPS (0-1000 nM, 2-fold dilutions) show binding to TM11 and PSGL-1 surfaces, as indicated by an increase in response units. The Kd values calculated by Biacore software are indicated in the figure. (C,D) Binding of increasing concentrations of PS (0-40 nM, 2-fold dilutions) show enhanced binding kinetics. (E,F) Binding of PS (20 nM) to TM11 and PSGL-1 was reduced in the presence of EDTA (10 mM). Residual binding of PS to TM11 in the presence of EDTA could be inhibited by coinjection of blocking antibody AK-4.
