Alexa Fluor Dye

The excitation and emission spectra of the Alexa Fluor series cover the visible spectrum and extend into the infrared.^[2] The individual members of the family are numbered according roughly to their excitation maxima (in nm).

Alexa Fluor dyes are synthesized through sulfonation of coumarin, rhodamine, xanthene(such as fluorescein), and cyanine dyes. Sulfonation makes Alexa Fluor dyes negatively charged and hydrophilic. Alexa Fluor dyes are generally more stable, brighter, and less pH-sensitive than common dyes (e.g. fluorescein, rhodamine) of comparable excitation and emission,^[3] and to some extent the newer cyanine series.^[4] However, they are also more expensive. They are patented by Invitrogen (which acquired the company that developed the Alexa dyes, Molecular Probes) and thus are priced higher than the common dyes that are available from multiple manufacturers.

Similar alternatives include the DyLight Fluors from Pierce (Thermo Fisher Scientific), and the Atto series from Atto-Tec and sold by Sigma-Aldrich

	Alexa Fluor		Abs/Em (nm)		Spectrally Similar Dyes		Note
	350™		346/442		AMC		The Alexa Fluor 350 dye is more water soluble than AMCA or AMCA-X and yields protein conjugates that are more fluorescent than those prepared from its nonsulfonated analog. Furthermore, its blueshifted spectra provide better separation from the fluorescence spectra of green fluorophores.
	405™		402/421		Pacific Blue dye, Cascade Blue dye		The Alexa Fluor 405 dye is the optimal fluorophore for 400–410 nm excitation sources, including the 405 nm spectral line of the blue-diode laser.
	430™		434/541
	488™		495/519		Fluorescein, Oregon Green 488 dye, Rhodamine Green dye, Cy2 dye		Alexa Fluor 488 protein conjugates are far superior to conjugates of fluorescein, Cy2, and any other green fluorophore, both in fluorescence intensity and photostability. Unlike fluorescein, the Alexa Fluor 488 dye is pH insensitive from pH 4 to 10.
	500™		502/525
	514™		517/542
	532™		532/554
	546™		556/573				TAMRA, CY3
	555™		555/565		Tetramethylrhodamine, Cy3 dye		Alexa Fluor 555 conjugates outperform conjugates of tetramethylrhodamine (TRITC and TAMRA) and Cy3.
	568™		578/603		Lissamine rhodamine B dye, Rhodamine Red dye		Alexa Fluor 568 conjugates are considerably brighter than Lissamine rhodamine B and Rhodamine Red-X conjugates and can be excited by the 568 nm spectral line of the Ar–Kr laser.
	594™		590/617		Texas Red dye, Cy3.5 dye		Alexa Fluor 594 conjugates are extremely photostable, more fluorescent than Texas Red conjugates and efficiently excited by the 594 nm spectral line of the orange He–Ne laser.
	610™		612/628
	633™		632/647
	647™		650/665		Cy5 dye, allophycocyanin (APC)		Total fluorescence of Alexa Fluor 647 secondary antibody conjugates is significantly higher than that of Cy5 conjugates supplied by other companies (Figure 3, page 27). Also, unlike the Cy5 dye, the Alexa Fluor 647 dye has very little change in absorption or fluorescence spectra when conjugated to most proteins, oligonucleotides, or nucleic acids, thus yielding greater total fluorescence at the same degree of labeling.
	660™		663/690		Cy5.5 dye, APC		The Alexa Fluor 660 dye exhibits good spectral separation from orange fluorophores, including the Alexa Fluor 555 dye and phycoerythrin (PE), making it a great choice for multicolor applications. The Alexa Fluor 660 dye is efficiently excited by the 633 nm and 647 nm spectral lines of the He–Ne laser and the krypton-ion laser, respectively.
	680™		679/702		NA		The Alexa Fluor 680 dye is highly recommended for use with the LI-COR Odyssey instrument for “In-Cell” Westerns.
	700™		702/723
	750™		749/775		Cy7 dye		The Alexa Fluor 750 dye is the longest-wavelength Alexa Fluor dye available.

Comparison with other dyes

The Alexa series dyes are less pH-sensitive and more photostable than the original dyes (fluorescein, rhodamine, etc.) from which they were synthesized.

However, brightness comparisons are not presently available. Brightness is commonly measured as a product of extinction coefficient (absorption efficiency) and quantum yield (emission efficiency) ^[5] While extinction coefficients are known (see the table above), the quantum yields of the Alexas have not been published by Molecular Probes.

In one case, a third party has compared one Alexa with another commonly used dye. This was a comparison of Cy5 and its Alexa with similar wavelength, Alexa 647, with the dyes conjugated to DNA. [1] This study found that Cy5 is brighter, but less photostable than Alexa 647.

Therefore presently it is difficult to quantitatively choose the best dye for a particular application and in many cases empirical testing is in order.

1. The Alexa Fluor Dye Series. Molecular Probes, Inc. (2006-04-06). Retrieved on 2007-08-13.
   3. Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, et al (1999). "Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates". J. Histochem. Cytochem. 47 (9): 1179-88. PMID 10449539.
2. Berlier JE, Rothe A, Buller G, et al (2003). "Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates". J. Histochem. Cytochem. 51 (12): 1699-712. PMID 14623938.
3. Souslova EA, Belousov VV, Lock JG, Stromblad S, Kasparov S, Bolshakov AP, Pinelis VG, Labas YA, Lukyanov S, Mayr LM, Chudakov DM (2007). "Single fluorescent protein-based Ca2+ sensors with increased dynamic range". BMC Biotechnol. 7 (7): 37. PMID 17603870.