Cyanines

Cyanine is the non-systematic name of a synthetic dye family belonging to the polymethine group. The word cyanin stems from the English word “cyan”, which conventionally means a shade of blue-green. Cyanine dyes exhibit large molar absorptivities (?150,000 to 300,000 M^-1cm^-1) and moderate quantum yields resulting in extremely bright fluorescence signals. Cyanines have proven useful in several fields including photography, biology, laser technology and analytical chemistry. AAT Bioquest offers the largest collection of cyanine dyes that maybe useful as molecular probes for a variety of biological applications. For certain biological applications, cyanines can advantageously replace other conventional dyes such as the fluoresceins and rhodamines.

Cyanine Dyes: Cy3 and Cy5

Among cyanine dyes, Cy3^® which fluoresces greenish yellow (?550 nm excitation, ?570 nm emission) and Cy5^® which is fluorescent in the red region (?650 nm excitation, ?670 nm emission) are the most popular. Because of their minimal spectral overlap, Cy3^®and Cy5^® are typically combined for 2 color detection. Cy3^® can be detected by various fluorometers, imagers, and microscopes with standard filters for TRITC. Due to its high molar extinction coefficient, Cy3^®dye is also easily detected by naked eye on electrophoresis gels, and in solution. Cy5^® became a popular replacement for far red fluorescent dyes because of its high extinction coefficient (as small as 1 nmol can be detected in gel electrophoresis by naked eye) and its fluorophore emission maximum in the red region, where many CCD detectors have maximum sensitivity and biological objects give low background interference.

Figure 2. Absorbance (dotted line) and emission (shaded region) maxima Cy3^®(left), Cy5^®(middle) and Cy7^®(right) dyes.

Spectral Properties

Table 1. Spectral Properties For Cyanine Dyes: Cy3 and Cy5

Cy Dye	Laser	Ex/Em (nm)	Filter Set	Extinction Coefficient (ε)1	Quantum Yield (Φ)2
Cy3	532 nm	554/568 nm	Cy3/TRITC	150,000	0.15
Cy5	633-647 nm	650/669 nm	Cy5	250,000	0.27
Cy7	730 nm	754/778 nm	Cy7	250,000	0.3

?: is the extinction coefficient (cm^-1M^-1).
?: is the fluorescence quantum yield.

Sulfonated vs Non-Sulfonated Cyanines

Sulfonated and non-sulfonated cyanine dyes exhibit very similar fluorescent characteristics and for many applications they can be used interchangeably. The difference between the dyes is mainly in their solubility. Non-sulfonated cyanines must be dissolved in an organic co-solvent, such as DMF or DMSO, prior to labeling of a biomolecule. While sulfonated cyanines can be used in aqueous conditions (the charged sulfonate groups decrease dye and conjugate aggregation). For purification by dialysis against water or an aqueous buffer, sulfonated cyanines must be used for efficient removal of any unreacted dye. Purification by gel filtration, chromatography, or electrophoresis is adequate for both sulfonated and non-sulfonated cyanines.

Table 2. Spectral comparison of sulfonated and non-sulfonated Cy3, Cy5 and Cy7 dyes.

	Cy3^®		Cy5^®		Cy7^®
Form	Sulfo	Non-Sulfo	Sulfo	Non-Sulfo	Sulfo	Non-sulfo
Ex/Em (nm)	554/568		650/669		754/778
Filter Set	Cy/TRITC		Cy5		Cy7
?	150,000	140,000	250,000	230,000	250,000	250,000
?	0.10	0.07	0.27	0.16	0.12	0.2
Cat No.	263	191	268	195	161	198

?: is the extinction coefficient (cm^-1M^-1).
?: is the fluorescence quantum yield.

Cyanine Products

We offer a broad selection of cyanine products including reactive dyes and conjugation kits for labeling biomolecules, conjugates, indicators and more.

Cyanine antibody and protein labeling kits Cy7, Cy3, Cy5
Annexin V conjugates for apoptosis detection Cy5.5, Cy3, Cy7, Cy5
Tandem dyes
Cyanine dyes for 2-D Fluorescence Difference Gel Electrophoresis (DIGE) Cy2DIGE, Cy3DIGE, Cy5DIGE
Cyanine labeled tyramide