Cy5 NHS Ester

Cy5® NHS Ester (GE Healthcare) and Cyanine5 NHS ester are chemically equivalent, far-red fluorescent dyes used for labeling peptides, proteins and oligonucleotides, with an excitation wavelength of 651 nm and an emission wavelength of 670 nm.

Background

Cy5 is a far-red fluorescent dye belonging to the cyanine dyes family, which contains other popular fluorophores such as Cy3 (yellow) and Cy7 (near infrared). Cy5 is sometimes also sold under the names, Cyanine5 or Sulfo-Cy5; these are all the same chemical structure.

The “sulfo” in the name derives from the SO₃^- groups present in the molecule, which improves its water solubility. Non-sulfonated Cy5, which lacks these groups, is less soluble and more likely to form aggregates in solution. Because of this, the sulfonated form of Cy3 is far more common than the non-sulfonated form.

Usage

Cy5 NHS ester has a molecular weight of 855.07 daltons. It is provided as a blue powder, with triethylammonium salts instead of potassium salts, for improved reconstitution in DMSO and DMF.

The NHS ester functional group enables easy conjugation to primary amines, such as the lysine residues of antibodies and amine-modified oligonucleotides. Cy5 is also available in maleimide form, for conjugation to the sulfide groups of proteins (e.g. cysteine), as well as phosphoramidite form, for usage in oligonucleotide synthesis.

Excitation and emission

Cy5 has an excitation peak at 651 nm and an emission peak at 670 nm. It is well excited by the 633 nm or 647 nm laser lines. For detection, a fluorescence channel such as the Cy5 filter set or a 660/20 bandpass filter can be used.

Applications

Cy5 can be conjugated to antibodies and used in flow cytometry. For example, Cy5-CD antibody conjugates are useful for the screening of cell surface markers and sorting by fluorescence (FACS). Cy5 can also be labeled to oligonucleotides, for use in real-time PCR techniques, such as in TaqMan probes.

Alternatives

Cy7 is a near-infrared alternative to Cy5 that has an excitation and emission of 756 nm and 779 nm, respectively.

iFluor 647 is a spectrally similar fluorophore that improves upon water solubility and brightness, while at a lower cost.