Figure 2. Direct binding of Ca²⁺ to N34–355.

(A) Recombinant N34–355 (3.6–6.9 μM per reaction) that had been purified in Ca²⁺-free buffer was incubated with increasing concentrations of ⁴⁵Ca²⁺ at known specific activities for 4 h at room temperature. Each sample was then passed over Ni-NTA beads to separate bound complexes from free Ca²⁺, and the radioactivity associated with N34–355 was measured by scintillation counting. The background-corrected radioactivity was used to calculate the amount of Ca²⁺ bound to the protein. The results shown are the means±S.E.M. for three experiments. (B) Scatchard plot of the binding results in (A).