Figure 1.

Identification of serum proteins that bind to recombinant CRASP-4. Recombinant, polyhistidine-tagged CRASP-4 was immobilized onto magnetic beads and incubated with NHS. Uncoated beads were also treated under the same conditions and used as a control to identify nonspecific binding of serum proteins. After extensive washing, bound proteins were eluted with 100mM glycine-HCl (pH 2.0) and the eluate fractions were separated by SDS-PAGE under nonreducing conditions. (a) Silver stain of a gel loaded with purified polyhistidine-tagged CRASP-4 (1 μg), eluate fraction of the uncoated beads, and the final wash and eluate fraction of CRASP-4-coated beads. (b) Western blot analysis of the eluate fraction of CRASP-4-coated beads using a polyclonal anti-CFH or a polyclonal anti-CFHR1 antiserum. Mobilities of molecular mass standards are indicated to the left.