Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye "Stains-all"

The Ca2+-binding proteins, calsequestrin, Calmodulin, troponin C, and S-100, have all been shown to stain dark blue or purple with the cationic carbocyanine dye "Stains-all", while most proteins stain red or pink. Stains-all staining of these Ca2+-binding proteins was 50-100% more intense than Coomassie blue staining, and the blue stained calsequestrin and troponin C could be detected in muscle extracts. Densitometric scans of Stains-all-stained gels revealed that interaction of the dye with Ca2+-binding proteins changed the absorption spectrum of the dye. The dye-protein complex absorbed maximally at 615 nm, reflecting a state that results from the binding of individual dye molecules at anionic sites. Stains-all also interacted with undenatured Ca2+-binding proteins in aqueous solution forming a complex absorbing maximally at 600 nm. A simple assay for Ca2+-binding proteins has been developed using this property. These results suggest that the interaction of the dye with anionic sites within these Ca2+-binding proteins produces the dye-protein complex which absorbs at 600-615 nm. Stains-all staining of these Ca2+-binding proteins will be useful in their identification and purification from various tissues. It might also be a valuable tool in the identification of potential Ca2+-binding proteins.