Identification of the cardiac sarcolemmal Na(+)-Ca2+ exchanger using monoclonal antibodies

Abstract

We have previously partially purified the sarcolemmal Na(+)-Ca2+ exchange protein and produced rabbit polyclonal antibodies to the exchanger (Philipson, K.D., Longoni, S., Ward, R. 1988. Biochim. Biophys. Acta 945:298-306). We now describe the generation of three stable murine hybridoma lines which secrete monoclonal antibodies (MAb's) to the exchanger. These MAb's immunoprecipitate 50-75% of solubilized Na(+)-Ca2+ exchange activity. The MAb's appear to be reactive with native conformation-dependent epitopes on the Na(+)-Ca2+ exchanger since they do not react on immunoblots. An indirect method was used to identify Na(+)-Ca2+ exchange proteins. A column containing Na(+)-Ca2+ exchanger immobilized by MAb's was used to affinity purify the rabbit polyclonal antibody. The affinity-purified polyclonal antibody reacted with proteins of apparent molecular weights of 70, 120, and 160 kDa on immunoblots of sarcolemma. The data provide strong support for our previous association of Na(+)-Ca2+ exchange with these proteins.