Fluorescence detection of calcium-binding proteins with quinoline Ca-indicator quin2

We have established a fluorescence method to detect calcium-binding proteins by making use of the quinoline Ca indicator quin2. Authentic calcium-binding proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred onto polyvinylidene difluoride membranes. Transfers were incubated with nonradioactive calcium ions, then with quin2 to detect the calcium-binding proteins as fluorescent bands by illumination with UV light. Calmodulin and parvalbumin of EF hand conformation calcium-binding type were clearly identified. Quin2 distinguished smooth muscle alpha-actinin from skeletal muscle alpha-actinin; the former was faintly fluorescent, having a low affinity for calcium ions. In whole myofibril preparations from skeletal muscles, troponin-C, connectin (titin), and nebulin were intensely fluorescent, being shown to have calcium-binding ability. The fluorescence method is an accurate, safe, and simple procedure to detect the binding of calcium ions to proteins following electrophoresis. The overlay technique described can be completed within 15 h and detects as little as 38 ng/well of troponin-C in the starting sample.