Fig. 1. Purification of Ca²⁺-binding proteins on phenyl-Sepharose monitored by a ⁴⁵Ca²⁺ blot overlay assay. Samples were subjected to SDS-PAGE, transferred to nitrocellulose and probed with ⁴⁵Ca²⁺ as described in the Materials and Methods. The free Ca²⁺ concentration was 1.4 µM. After autoradiography (B), the membrane was stained with amido black (A). Lane 1, markers, 2 µg each; lane 2, phenyl-Sepharose load, 50 µg; lane 3, phenyl-Sepharose EGTA eluate, 20 µg; M, M_r of markers (x10³).