Jackson ImmunoResearch Europe, Ltd

Technical Information on Probes
Conjugated to Affinity-Purified
Antibodies and to Other Proteins:

Cyanine Dyes (Cy2, Cy3, and Cy5)

Technical Service e-mail
tech@jireurope.com tech@jacksonimmuno.com

Cy2 conjugates have maximum adsorption/excitation around 492 nm and fluoresce at 510 nm in the green region of the visible spectrum (Table 1and Figure 1) like FITC (520 nm), but they are more photostable, less sensitive to pH changes, and more fluorescent in organic mounting media than FITC. However, we now recommend DyLight 488 as the preferred green-fluorescing dye because it is brighter and more photostable than Cy2 and FITC. The same filters used for Cy2 and FITC are appropriate for use with DyLight 488. A further disadvantage of Cy2 is its sensitivity to p-phenylenediamine, an anti-fading agent found in some commercial mounting media, which results in weak and diffused fluorescence after storage of stained slides.

Cy3 is brighter, more photostable, and gives less background than TRITC and most other fluorophores, with the exception of DyLight 549, which is as bright and somewhat more photostable. Cy3 conjugates can be excited maximally at 550 nm, with peak emission at 570 nm (Table 1 and Figure 1). For fluorescence microscopy, Cy3 can be visualized with traditional tetramethyl rhodamine (TRITC) filter sets since the excitation and emission spectra (Figure 1) are nearly identical to those of TRITC.

Table 1. Approximate peak wavelengths of absorption and emission for different fluorophore-conjugated, affinity-purified antibodies. Approximate values are given for purposes of comparing one fluorophore with another. Actual values may vary depending on the spectrofluorometer used in each laboratory.

Fluorophore	Excitation Peak (nm)	Emission Peak (nm)
DyLight 405	400	421
Aminomethylcoumarin, AMCA	350	450
Cyanine, Cy2	492	510
DyLight 488	493	518
Fluorescein, FITC	492	520
DyLight 549	555	568
Indocarbocyanine, Cy3	550	570
Tetramethyl Rhodamine, TRITC	550	570
Rhodamine Red-X, RRX	570	590
DyLight 594	591	616
Texas Red, TR	596	620
Indodicarbocyanine, Cy5	650	670
DyLight 649	652	670

Excitation	Emission

Figure 1. Excitation and emission spectra of different fluorophore conjugated, affinity-purified antibodies. This figure illustrates only the relative shape and position of each fluorophore in the peak region of its excitation and emission following conjugation to antibodies. Quantitative comparisons should not be made since peak heights have been normalized. For clarity, spectra for DyLight-antibody conjugates are not included here. For comparison, see DyLight-antibody spectra. All spectra were obtained with a M-Series spectrofluorometer system from Photon Technology International, Inc.

Cy3 can be excited to about 50% of maximum with an argon laser (514 nm or 528 nm lines), or to about 75% of maximum with a helium/neon laser (543 nm line) or mercury lamp (546 nm line). Cy3 has been used with fluorescein for double labeling. However, the use of a narrow band-pass emission filter for fluorescein is recommended to minimize Cy3 fluorescence in the FITC filter set. Cy3 can also be paired with Cy5 for multiple labeling when using a confocal microscope.

Cy5 conjugates are excited maximally at 650 nm and fluoresce maximally at 670 nm (Table 1 and Figure 2). They can be excited to about 98% of maximum with a krypton/argon laser (647 nm line) or to about 63% of maximum with a helium/neon laser (633 nm line). Cy5 can be used with a variety of other fluorophores for multiple labeling due to a wide separation of its emission from that of shorter wavelength-emitting fluorophores. However, we now recommend DyLight 649 as the preferred far-red-fluorescing dye because it is brighter than Cy5. A significant advantage of using Cy5 and DyLight 649 over other fluorophores is the low autofluorescence of biological specimens in this region of the spectrum. However, because of their emission maximum at 670 nm, they cannot be seen well by eye, and they cannot be excited optimally with a mercury lamp. Therefore, they are not recommended for use with conventional epifluorescence microscopes. They are most commonly visualized with a confocal microscope equipped with an appropriate laser for excitation and a far-red detector. Cy5 and DyLight 649 conjugates are a less expensive and equally bright alternative to Allophycocyanin conjugates for flow cytometry.

Although anti-fading agents usually are not required when visualizing cyanine dye conjugates in an epifluorescence microscope, anti-fading agents should be added to aqueous mounting media for confocal laser scanning microscopy. For specimens labeled with Cy2, avoid the use of mounting media containing p-phenylenediamine, since this anti-fading agent reacts with Cy2, resulting in weak and diffused fluorescence after storage of stained slides. Other anti-fading agents, such as n-propyl gallate, may be used for mounting cyanine dye stained sections in aqueous media. Organic based mounting media, such as DPX or methyl salicylate, also may be used with cyanine dyes.

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