Cy5

Cy5 is a fluorescent cyanine dye used as a label in molecular biology and related fields. It belongs to the cyanine dye family and is typically a water-soluble pentamethine dye with sulfonate groups that improve aqueous solubility and reduce nonspecific binding. Cy5 is commonly employed as a tagging molecule for nucleic acids, proteins, and antibodies.

Spectral properties of Cy5 place it in the far-red region of the spectrum. Its absorption maximum is around 650 nm, with excitation near 649–650 nm and emission around 670–675 nm. This long-wavelength emission helps minimize autofluorescence from biological samples and is compatible with common red and near-infrared laser lines. The dye's brightness and performance depend on the chemical form used (such as sulfo-Cy5 derivatives) and the labeling context.

Cy5 is widely used in fluorescence labeling and detection techniques, including fluorescence in situ hybridization (FISH), DNA microarrays, immunofluorescence, and flow cytometry. It is also employed in various nucleic acid probes and detection reagents, where its far-red emission reduces background signals when paired with other dyes.

Chemically, Cy5 is often utilized in derivative forms that facilitate conjugation, such as NHS ester or sulfonated variants, enabling attachment to amines or other targets. Conjugates must be compatible with the imaging system, and appropriate filter sets and detectors should be selected to match its emission.

Limitations and practical considerations include potential photobleaching under intense illumination and spectral overlap with related dyes like Cy5.5 or Alexa Fluor 647, which can require careful spectral unmixing or filter selection. Storage typically involves protecting the dye from light and storing at low temperatures to preserve stability.