FIG. 6.

Analysis of binding of various mutants to Lf- and Tf-HRP conjugates. Equal amounts of lysed cell suspension obtained from bacteria grown under iron-restricted conditions were spotted directly onto nitrocellulose membranes (in triplicate). After blocking, the blots were incubated in either high-stringency Tf-binding buffer (50 mM Tris, 1 M NaCl [pH 8.0]), high-stringency Lf-binding buffer (50 mM Tris, 1 M NaCl [pH 9.0]), or low-stringency Lf-binding buffer (50 mM Tris, 0.1 M NaCl [pH 6.0]), containing 1:1,000 dilutions of peroxidase-conjugated human Lf (hLf-HRP) or human Tf (hTf-HRP). After repeatedly being washed in the binding buffer, the blots were subsequently developed. The N. meningitidis TbpA⁻ and TbpB⁻ mutants used as controls have been characterized previously (24).