Fig. 5.

pH dependence of ligand binding to human mincle. Binding of ¹²⁵I-mannose-BSA to the biotin-tagged CRD immobilized on streptavidin-coated wells was measured in buffers containing 150 mM NaCl, 3 mM CaCl₂, 25 mM 2-(N-morpholino)ethanesulfonic acid and 25 mM 3-(N-morpholino)propanesulfonic acid at various pH values. Following incubation with ligand for 2 h at 4°C, plates were washed 3× with binding buffer containing 150 mM NaCl, 3 mM CaCl₂ and 25 mM Tris–Cl, pH 7.8. The bound radioactivity was normalized to the maximal binding observed at pH 7.7–8.1.