| Non- sulfonated cyanines - fluorescent dyes There are two varieties of cyanine dyes: non- sulfonated cyanines, and sulfonated cyanines. For many applications they are interchangeable, because their spectral properties are nearly identical. Both sulfonated and non-sulfonated dyes can be used for the labeling of biomolecules such as DNA and proteins. The difference between the dyes is their solubility: sulfo- dyes are water-soluble, and they do not use of organic co-solvent for the labeling in aqueous environment. They are less prone to aggregation in water. There are cases when one of the type of cyanines is desired. Available non-sulfonated dyes incude Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5.
Cy stands for 'cyanine', and first digit is number of carbon atoms between indolenine groups. Cy2 which is oxazole derivative rather than indolenin, is an exception from this rule. Suffix .5 is added for benzo-fused cyanines. Variation of the structures allows to change fluorescence properties of the molecules, and to cover most important part ot visible and NIR spectrum with several fluorophores.
Most derivatives of non-sulfonated cyanines (except for hydrochlorides of hydrazides and amines) have low aqueous solubility. When these molecules are used for biomolecule labeling, use of organic co-solvent (5-20% of DMF or DMSO) is necessary for efficient reaction. Cyanine dye should be dissolved in organic solvent first, and added to a solution of biomolecule (protein, peptide, amino- labeled DNA) in appropriate aqueous buffer. When conjugation takes place efficiently, the dye reacts before it precipitates. Fluorescent properties of non-sulfonated cyanines have little dependence on solvent and surrounding. Absorbance and fluorescence spectra of non-sulfonated cyanine dyes are plotted below. 
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