SPR analysis of the binding of IF-cobalamin to human cubilin. Human cubilin was immobilized to a sensor chip, and the on and off rates for ligand binding were recorded on a BIAcore 2000. The arrows indicate the start of flow with ligand (association phase) and the start of wash with buffer alone (dissociation phase). The recorded sensorgrams in the upper panel show binding of 400 nmol/L porcine intrinsic factor in normal flow buffer with 0.5 mmol/L free Ca²⁺ and the absence of binding when 5 mmol/L EDTA was added to the flow buffer. The lower panel shows binding activity of diluted porcine gastric mucosa extract (114 μg protein/mL) with and without saturation with cobalamin. The sensorgrams display the values after subtraction of the values from the same recordings on a blank chip.