Flagella were prepared as described previously (Totten and Lory, 1990). Bacteria were collected by centrifugation (7000 g, 10 min), resuspended in 50 mM Na-phosphate (pH 7.0), 10 mM MgCl2, and treated in a Wareing blender for 60 sec to shear off the flagella. Cells and cell debris were removed by low speed centrifugation (60 min at 10 000 g), and flagella were collected by high-speed centrifugation (60 min at 100 000 g). The pellet was resuspended in H2O and the pH was lowered to 2.0 by glycine-HCl to dissociate the flagella. After removal of insoluble material by centrifugation
(60 min at 100 000 g), the supernatant containing the dissociated flagellin molecules was adjusted to pH 7.0 with NaOH.