Stains-All is a cationic dye of carbocyanine.

Instructions
1. Preparation of stock solution and working solution: Stains-All can be dissolved in deionized water or organic solvents (such as ethanol); it is usually prepared at a working concentration of 0.5–5 µM, depending on the experimental conditions.
2. Staining steps:
1) Dissolve the target protein (such as calmodulin) in an appropriate buffer (such as PBS, pH 7.4).
2) Add an appropriate amount of Stains-All to the protein solution and mix gently.
3) Incubate at room temperature for 10–30 minutes to allow the dye to bind to the calcium binding site.
3. Fluorescence detection: Use a fluorescence spectrophotometer to excite at 630 nm and usually measure the emission spectrum at around 650 nm.
4. Data analysis: Compare the changes in fluorescence signals under different calcium concentrations to analyze the binding kinetics of calcium and calmodulin; in the titration experiment, the fluorescence response curve is drawn by gradually increasing the concentration of calcium ions, and then the binding constant is calculated. "