Cyanine3 NHS Ester

Cy3® NHS Ester (GE Healthcare) and Cyanine3 monosuccinimidyl ester are chemically equivalent, reactive dyes used for fluorescence labeling biological molecules by targeting amino-groups.

Background

Cy3 is a yellow fluorescent dye belonging to the cyanine dyes family. It is sometimes also referred to as Sulfo-Cy3; the two are chemically equivalent. In contrast, non-sulfonated Cy3 does not have SO₃^- groups. Consequently, non-sulfonated Cy3 has lower water solubility than Cy3, as well as increased likelihood of forming aggregates in squeous solution. For this reason, sulfo-Cy3 and Cy3 are much more common than non-sulfonated Cy3.

Usage

Cy3 NHS ester has a molecular weight of 829.03 daltons. It is supplied as a red powder and can be reconstituted using DMSO. The NHS ester functional group allows for easy conjugation to targets with primary amine groups, such as the lysine residue of proteins and antibodies or amine-modified oligonucleotides. Labeling reactions create a covalent bond between the dye and biomolecule; for the best efficiency, the reaction should occur in a buffer with pH = 8.5 ± 0.5. Once reacted, a degree of labeling (DOL) should be calculated for the conjugate. This is important as overlabeling (i.e. high DOL) can result in self-quenching of the Cy3 fluorohpore.

Excitation / Emission

Cy3 has two important wavelengths: an excitation peak at 555 nm and an emission peak at 569 nm. When excited by a suitable light source, such as a 532 nm or 555 nm laser, it will emit yellow fluorescence. As such, compatible fluorescence channels include Cy3 or TRITC filter sets. Cy3 fluorescence is fairly resistant to changes in pH, tolerating pH 4 to pH 10.

Applications

Cy3 can be conjugated to antibodies and used for immunofluorescence (IF) and immunocytochemistry (ICC) applications. For example, cells treated with mouse anti-tubulin primary antibodies, followed by Cy3 goat anti-mouse IgG secondary antibodies, can be used to visualize cytoskeletal structures.

A second application is the use of Cy3 to label oligonucleotides. In fluorescence in situ hybridization (FISH), Cy3-oligo probes allow for spatial mapping of genetic material in cells. Combined with a quencher, Cy3-oligo conjugates can be used as TaqMan probes (or molecular beacons) for real-time PCR techniques.

Other formats

Cy3 is also available with maleimide moieties. This allows for conjugation to proteins and antibodies which present sulfhydryl-groups, such as cysteine residues. For incorporation of Cy3 into an oligonucleotide during chemical synthesis, Cy3 phosphoramidites can be used. In this process, phosphoramidites are added from the 3' to 5' direction.

Cy5 is an alternative to Cy3 that belongs to the same family for fluorescent dyes. It has an excitation wavelength of 651 nm and an emission wavelength of 670 nm.

Cy3B is an analog of Cy3 that is more resistant to internal de-excitation, thus resulting in a measured increase in fluorescence signal and improved photostability.

iFluor 555 is a spectrally similar fluorophore that offers improved water solubility and brightness, with decreased photobleaching and cost.