The Alexa Fluor Dye Series — Peak Performance across the Visible Spectrum
The Alexa Fluor dyes — a series of superior fluorescent dyes that span the visible spectrum — represent a major breakthrough in the development of fluorescent labeling reagents1,2, especially when combined with the multi-purpose Zenon technology (Section 7.3 MP catalog) and nucleic acid detection methods (Section 8.5 MP catalog). Download the Alexa Brochure (PDF, 1.38 MB) Antibody labeling kits from A(lexa Fluor®) to Z(enon®) (PDF, 1.75 MB)		Alexa Fluor 350 Dye Alexa Fluor 405 Dye Alexa Fluor 430 Dye Alexa Fluor 488 Dye Alexa Fluor 500 Dye Alexa Fluor 514 Dye Alexa Fluor 532 Dye Alexa Fluor 546 Dye Alexa Fluor 555 Dye Alexa Fluor 568 Dye Alexa Fluor 594 Dye Alexa Fluor 610 Dye Alexa Fluor 633 Dye Alexa Fluor 647 Dye Alexa Fluor 660 Dye Alexa Fluor 680 Dye Alexa Fluor 700 Dye Alexa Fluor 750 Dye
		Figure 1. Please click to enlarge and for a description
Benefits of the Alexa Fluor dyes and their conjugates include: Brightness — Alexa Fluor conjugates exhibit more intense fluorescence than other spectrally similar conjugates Photostability — Alexa Fluor conjugates are more photostable than most other fluorescent conjugates, allowing more time for image capture (pict., pict. ) Instrument compatibility — Absorption spectra of the Alexa Fluor conjugates are matched to the principal output wavelengths of common excitation sources Color selection — Alexa Fluor conjugates are available in several distinct fluorescent colors, ranging from blue to red pH insensitivity — Alexa Fluor dyes remain highly fluorescent over a broad pH range Water solubility — Alexa Fluor reactive dyes have good water solubility, so protein conjugations can be performed without organic solvents, and the conjugates are relatively resistant to precipitation during storage
Figure 2. Emission spectra for the Alexa Fluor dye series.

Alexa Fluor 350 Dye — Bright Blue and UV Light–Excitable

The blue-fluorescent Alexa Fluor 350 dye produces conjugates that are typically greater than 50% more fluorescent than conjugates prepared from AMCA (pict.). Furthermore, because Alexa Fluor 350 conjugates have slightly shorter-wavelength emission maxima than AMCA conjugates (442 nm versus 448 nm), the fluorescence of Alexa Fluor 350 conjugates is better separated from that of commonly used green fluorophores (pict., ).

Alexa Fluor 405 Dye — A Near-Perfect Match to the Blue Laser Diode

With excitation/emission maxima of 402/421 nm, the Alexa Fluor 405 dye () is a near-perfect match to the 405 nm spectral line of the blue diode laser recently developed for fluorescence microscopy and flow cytometry. The Alexa Fluor 405 succinimidyl ester is an amine-reactive derivative of the Cascade Blue dye, which was previously available in amine-reactive form only as an acetyl azide. Not only is it offered at higher purity than is Cascade Blue acetyl azide, but the Alexa Fluor 405 succinimidyl ester also contains a 4-piperidinecarboxylic acid spacer that separates the fluorophore from its reactive moiety, minimizing any interactions between the fluorophore and the biomolecule to which it is conjugated.

Alexa Fluor 430 Dye — Absorption at 430 nm with a High Stokes Shift

Few reactive dyes that absorb between 400 nm and 450 nm have appreciable fluorescence beyond 500 nm in aqueous solution. The Alexa Fluor 430 dye fills this spectral gap. Excitation near its absorption maximum at ~430 nm is accompanied by strong emission near 540 nm (, pict.).

Alexa Fluor 488 Dye — The Best Green Fluorophore

Protein conjugates prepared with the Alexa Fluor 488 dye are far superior to conjugates of fluorescein, and of any other green fluorophore that we have tested, including those of the Cy2 dye (pict.). Not only are Alexa Fluor 488 conjugates significantly brighter than fluorescein conjugates (pict), they are much more photostable (pict., pict., pict., pict.). Furthermore, fluorescence of the Alexa Fluor 488 fluorophore is independent of pH from 4 to 10. This pH insensitivity is a major improvement over fluorescein, which emits fluorescence that is significantly affected by pH (pict., pict. , ).

Alexa Fluor 500 and Alexa Fluor 514 Dyes — Two Green Fluorophores for Optical Separation Systems

The green-fluorescent Alexa Fluor 500 and Alexa Fluor 514 dyes are specifically designed to be detected simultaneously with other green fluorophores, despite their spectral overlap (, ). Though they appear similar in color by eye, the Alexa Fluor 500 dye can be optically separated from the Alexa Fluor 514 dye using the Zeiss META system or similar spectral imaging instruments with linear-unmixing software; these instruments have the capability of differentiating between fluorescence emission maxima <5 nm apart. Similarly, the fluorescent signal from the Alexa Fluor 514 dye can be resolved from both the Alexa Fluor 488 and the Alexa Fluor 500 fluorescence emissions. Furthermore, the photostable and pH-insensitive Alexa Fluor 514 dye is probably the best fluorophore available for the 514 nm spectral line of the argon-ion laser.

Alexa Fluor 532 Dye — The Optimal Dye for 532 nm Excitation Sources

With excitation and emission spectra intermediate between those of the green-fluorescent Alexa Fluor 488 dye and orange-fluorescent Alexa Fluor 546 dye (), the Alexa Fluor 532 dye and its conjugates are ideal for use with 532 nm excitation sources, including the frequency-doubled Nd:YAG laser (pict).

Alexa Fluor 546 Dye — A More Fluorescent Alternative to Cy3 and Tetramethylrhodamine

Conjugates prepared with the Alexa Fluor 546 dye are perfect for applications that require fluorescent probes that emit in the orange region of the spectrum. These intensely fluorescent conjugates outperform conjugates of tetramethylrhodamine (TRITC and TAMRA) and Cy3 (Fig, pict) and are readily excited by the strong 546 nm emission of mercury-arc lamps (, pict).

Alexa Fluor 555 Dye — A Superior Alternative to the Cy3 Dye

Spectra of the Alexa Fluor 555 conjugates virtually match those of the Cy3 dye (Fig, ), resulting in an optimal match to filters designed for that dye. However, total fluorescence of Alexa Fluor 555 conjugates is higher (Fig, Fig). The Alexa Fluor 555 dye is also more photostable than Cy3 (Fig), providing researchers with additional time for image capture.³

Alexa Fluor 568 Dye — Perfect for 568 nm Excitation Sources

The red-orange–fluorescent Alexa Fluor 568 dye is optimally excited by the 568 nm spectral line of the Ar–Kr mixed-gas laser used in many confocal laser-scanning microscopes. Alexa Fluor 568 conjugates are considerably brighter than Lissamine Rhodamine B conjugates or even Rhodamine Red-X conjugates, which have similar excitation and emission maxima (pict, ).

Alexa Fluor 594 Dye — A Superior Alternative to the Texas Red Dye

Conjugates prepared from the Alexa Fluor 594 dye emit in the red region of the spectrum (), making them particularly useful for multilabeling experiments in combination with green-fluorescent probes. Alexa Fluor 594 conjugates are much more fluorescent than are Texas Red conjugates (Fig, pict).

Alexa Fluor 610 Dye — The Red Jewel of the Alexa Fluor Dyes

The bright and photostable Alexa Fluor 610 dye emits an intense red fluorescence that can be visualized with the same optics used for the Texas Red and Alexa Fluor 594 dyes. With excitation/emission maxima of 612/628 nm (), the Alexa Fluor 610 dye can be easily differentiated from green fluorophores, making it an ideal candidate for multicolor labeling. Unlike the fluorescence of the Alexa Fluor 633 dye and longer-wavelength fluorophores, fluorescence of the Alexa Fluor 610 dye can still be seen with the human eye. The Alexa Fluor 610 dye is recommended to be utilized to prepare tandem conjugates of phycobiliproteins with improved spectral properties.

Alexa Fluor 633 Dye — The Optimal Dye for the 633 nm He–Ne Laser Line

Far-red–fluorescent dyes are among the most sought-after labels for fluorescence imaging because their spectra are well beyond the range of most sample autofluorescence. The growing popularity of the 633 nm spectral line of the He–Ne laser and the 635 nm spectral line of red diode lasers prompted us to create compatible dyes. Although their fluorescence is not visible to the human eye, Alexa Fluor 633 conjugates are bright and photostable, with peak absorption centered at 632 nm and a peak emission at 650 nm ⁴ ().

Alexa Fluor 647 Dye — A Superior Alternative to the Cy5 Dye

Spectra of the Alexa Fluor 647 conjugates virtually match those of the Cy5 dye (Fig), resulting in an optimal match to optical filters designed for that dye. However, total fluorescence of the secondary antibody conjugates of the Alexa Fluor 647 dye is significantly higher than that of Cy5 conjugates supplied by other companies (Fig, Fig, Fig). Also, unlike the Cy5 dye, the Alexa Fluor 647 dye has very little change in absorbance or fluorescence spectra when conjugated to most proteins, oligonucleotides and nucleic acids (Fig), thus yielding greater total fluorescence at the same degree of substitution.⁵

Alexa Fluor 660 Dye — An Excellent Dye for the 647 nm Krypton-Ion Laser Line

The Alexa Fluor 660 dye is optimally excited by the 647 nm spectral line of the krypton-ion laser and well excited by the 633 nm spectral line of the He–Ne laser. Protein conjugates of the Alexa Fluor 660 dye produce bright far-red–fluorescence emission, with a peak at 690 nm. The wide separation of its emission from that of other fluorophores allows use of the Alexa Fluor 660 dye with other fluorescent labels, including the Alexa Fluor 546 and Cy3 dyes and phycoerythrin conjugates (). The Alexa Fluor 660 dye is the dye of choice as a "second label" with allophycocyanin (APC) conjugates in flow cytometry applications.

Alexa Fluor 680 Dye — An Alternative to the Cy5.5 Dye

With a peak excitation at 679 nm and maximum emission at 702 nm, the Alexa Fluor 680 dye is spectrally similar to the Cy5.5 dye (Fig). Fluorescence emission of the Alexa Fluor 680 dye is well separated from that of other commonly used red fluorophores, such as the tetramethylrhodamine, Texas Red, R-phycoerythrin, Alexa Fluor 594 and Alexa Fluor 647 dyes, making it ideal for three- and four-color labeling ⁶ ().

Alexa Fluor 700 Dye — The Optimal Dye for Far-Red Diode Lasers

With an absorption maximum at 696 nm, the Alexa Fluor 700 dye can be excited with a xenon-arc lamp, far-red diode lasers or dye-pumped lasers operating in the 675–700 nm range. The Alexa Fluor 700 dye provides infrared fluorescence emission, with a peak at 719 nm ().

Alexa Fluor 750 Dye — The Longest-Wavelength Alexa Fluor Dye

Spectrally similar to the Cy7 dye (Fig), the Alexa Fluor 750 dye is the longest-wavelength Alexa Fluor dye currently available. Its fluorescence emission maximum at 779 nm is well separated from commonly used far-red fluorophores such as Alexa Fluor 647, Alexa Fluor 660 or allophycocyanin (APC), facilitating multicolor analysis. With a peak excitation at ~752 nm, conjugates of the Alexa Fluor 700 dye are well excited by a xenon-arc lamp or dye-pumped lasers operating in the 720–750 nm range ().

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