Identification of the cardiac sarcolemmal Na+−Ca2+ exchanger using monoclonal antibodies

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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 expitopes 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 proteinsof, apparent molecular weights of 70, 120, and 160 kDa on immunoblots of sarcolemma. The data provide strong support for our prevous association of Na+−Ca2+ exchange with these proteins.