FLUORESCENCE DETECTION OF CALCIUM-BINDING PROTEINS WITH QUINOLINE CA-INDICATOR QUIN2

We have established a fluorescence method to detect calcium-binding pr oteins by making use of the quinoline Ca indicator quin2. Authentic ca lcium-binding proteins were subjected to sodium dodecyl sulfate-polyac rylamide gel electrophoresis and then electrophoretically transferred onto polyvinylidene difluoride membranes. Transfers were incubated wit h nonradioactive calcium ions, then with quin2 to detect the calcium-b inding proteins as fluorescent bands by illumination with UV light. Ca lmodulin and parvalbumin of EF hand conformation calcium-binding type were clearly identified. Quin2 distinguished smooth muscle alpha-actin in from skeletal muscle alpha-actinin; the former was faintly fluoresc ent, having a low affinity for calcium ions, In whole myofibril prepar ations from skeletal muscles, troponin-C, connectin (titin), and nebul in were intensely fluorescent, being shown to have calcium-binding abi lity. The fluorescence method is an accurate, safe, and simple procedu re to detect the binding of calcium ions to proteins following electro phoresis, The overlay technique described can be completed within 15 h and detects as little as 38 ng/well of troponin-C in the starting sam ple. (C) 1997 Academic Press.