Cy5 carboxylic acid is a non-activated carboxylic acid with low aqueous solubility. For coupling with amines, or protein labeling, Cy5 NHS ester and water-soluble sulfo-Cy5 NHS ester is preferred. This labeling dye has limited water solubility. For biomolecule labeling, the labeling reagent has low aqueous solubility, using of organic co-solvent to dissolve this molecular is necessary for efficient reaction. First, Cyanine dye should be dissolved in organic solvent and then added to a solution of biomolecule in appropriate aqueous buffer.
