Routine detection of calcium-binding proteins following their adsorption to nitrocellulose membrane filters

A routine semiquantitative procedure which permits soluble calcium-binding proteins to be detected following their adsorption to nitrocellulose membrane filters by liquid scintillation counting of specifically bound 45Ca is described. Proteins with high affinity for calcium such as calmodulin and troponin can be detected with a detection threshold of about 2 micrograms per 400 microliter. Modifications to decrease this limit are feasible and are discussed. This technique should allow calcium-binding proteins of unknown function to be assayed during their purification. It was necessary to treat solutions containing 45Ca with chelex-100 in order to prevent loss of calcium binding which occurred as the decay product (Sc3+) accumulated, suggesting that all studies utilizing 45Ca as a tracer should evaluate possible interference by this ion.