Concentration dependence of α-adducin tail binding to immobilized cdb3. Cytoplasmic domain of band 3, ovalbumin, or no protein was reacted with Affi-Gel 15 beads, as described in “Binding of α-adducin tail to cdb3 immobilized on Affi-Gel 15.” Different concentrations of His-tagged α-adducin tail were incubated with the protein-derivatized beads for 4 hours at 4°C with gentle shaking. Beads were pelleted, washed 3 times with PBS, eluted, separated electrophoretically by SDS-PAGE, and analyzed by Western blotting using anti–α-adducin antibody (A). Quantitative densitometry was performed using Image J, and the resulting data were fit to a noncooperative single site-binding equilibrium, which yielded an apparent K_D of 35 ± 7 nM (B). A.U. indicates arbitrary units.