Calcimedins: Purification and production of antibodies

This chapter discusses the purification of calcimedins from chicken gizzard, bovine liver, and rat liver. The procedure for calcimedin purification from chicken gizzard is modified for application to the bovine liver. Antibodies were raised in sheep using rat 67K and 35K calcimedins. Sheep were injected with an initial dose of 7 mg of protein followed by subsequent injections of 2 mg protein on days 30, 44, and 58. Antibodies were affinity purified on corresponding calcimedin bound to Sepharose columns. Purified antibodies were dialyzed against 100 mM sodium borate buffer (pH 8.4) containing 75 mM NaCl. They were stored in the same buffer. Aliquots from rat liver homogenates and in the presence of Ca²⁺ phenyl-Sepharose-purified samples were subjected to SDSPAGE on 15% gels. The separated proteins were electrophoretically transferred to nitrocellulose. Antibody to 35K calcimedin was tested against all proteins. Antibody reacted with both 67K and 35K calcimedins in phenyl-Sepharose-purified samples. In addition, the anti-35K antibody showed cross-reactivity with two more proteins (MW 47K and MW 27K) in the cytosol. These results suggest that the 35K and 67K calcimedins have some common epitopes. Moreover, the immunoreactive 47K and 27K species may also share common epitopes with the 35K calcimedin.