Fluorescent Compounds

Research Organics offers a wide array of fluorescent labels, dyes and derivatizing reagents for use in fluorescence microscopy, immunochemistry and flow cytometry. Cy3, Cy5, FluorX and Fluorolink reagents represent a new generation of fluorescent label compounds for proteins, modified oligonucleotides and other compounds containing primary amine groups. These cyanine dyes, offered in convenient kit forms, produce conjugates that are much brighter, more photostable and emit less background than most other fluorophores.

Conjugations proceed smoothly and quickly at room temperature. The dyes are also water soluble, eliminating the need for organic solvents and for producing conjugates that remain in solution (1,2).

FluorX is a new reactive fluorescent dye kit that produces stable, high purity fluorescein- based conjugates. FluorX is the N-hydroxy succinimidyl (NHS) ester of carboxyfluorescein, which contains an extended linker arm. This compound binds covalently with primary aliphatic amine groups under mild reaction conditions to form stable fluorescent analogues of biomolecules, such as antibodies, proteins and amine-modified oligonucleotides. The linker arm reduces steric hinderance, permitting greater access to primary amine groups recessed in globular regions of proteins and other biomolecules.

Compounds labeled with the FluorX dye have an absorption maximum of 494 nm and an emission maximum of 520 nm, which can be easily detected by fluorescence microscopy using standard fluorescein filter sets. The FluorX Dye is ideally excited by the 488 nm line of an argon laser and efficiently excited by sources such as mercury or xenon arc lamps. The conjugates produce an intense yellow-green fluorescence, making the signal visually detectable and distinguishable from other fluorescent labels such as TRITC, XRITC, Cy3 or Cy5.

Cy3 has an emission maximum at 565 nm and an absorption at 550 nm. It can be excited up to 50 percent maximum using an Argon laser (514 nm) and to 75 percent maximum using a He/Ne laser (543 nm) or a Mercury lamp (546 nm). For light microscopy, Cy3-labeled samples can be observed using standard optical filters for tetramethylrhodamine preparations (TRITC) because of their similar spectral properties. This also makes Cy3 suitable for double labeling applications with fluorescein-tagged compounds such as FluorX, FITC or DTAF. Biomolecules labeled with Cy3 have been shown to display superior brightness in fluorescence microscopy applications compared to standard fluorescent labels such as Texas Red and TRITC (3).

Cy5 has an emission maximum at 667 nm and an absorption maximum at 652 nm, with an extremely high extinction coefficient of >200,000 M-1 cm-1 Cy5-labeled compounds are easily detected at this high emission wavelength by far-red photo-imaging systems, especially CCD cameras. The dye can be excited with a Krypton /Argon laser (647 nm) or excited to 63% of maximum with He/Ne laser at 633 nm. Mercury or Tungsten-Halogen lamps also serve as suitable excitation sources.

Cy5 can be used for multiple labeling applications because of a wide spectral separation of its emission from other fluorescent labels, including Cy3. A benefit of Cy5 is the low autofluorescence obtained from biological samples at the excitation wavelength the dye requires.

Cy3 and Cy5 can be used together with FluorX in multiple labeling experiments. The similarities in the reactive NHS functional groups of the FluorX dye and the Cy3/Cy5 dyes (as shown in the structural formulas on page) allow conjugation to biological compounds to proceed under the same mild reaction conditions.

Research Organics, Inc., 4353 East 49th St., Cleveland, OH 44125. Tel: 216-883-8025; Fax: 216-883-1576.