Alexa Fluor

Alexa Fluor is a family of fluorescent dyes developed by Thermofisher Scientific. They have the properties of high brightness, photostability, and low pH sensitivity. Primarily, these fluorophores are used in biological assays and imaging techniques, including fluorescence microscopy, flow cytometry, and immunofluorescence. In these applications, Alexa Fluors are often conjugated (e.g. covalently bound) to biomolecules such as proteins, antibodies, and nucleic acids, enabling their detection and visualization in fluorescence-based assays. (Panchuk et al., 1999; Rasmussen et al., 2008)

The Alexa Fluor series of dyes exhibit a broad range of excitation and emission spectra covering the visible spectrum and extending into the infrared. They are brighter and more photostable than classic dyes such as FITC (Fluorescein isothiocyanate), making them more suitable for use in long exposure imaging and applications where high signal intensity is required. However, high background signals may arise due to potential quenching or autofluorescence by other compounds in the matrix, and issues with fluorescence scattering. (Lutea et al., 2005)

The number suffixed to each Alexa Fluor dye in the series indicates its optimal excitation wavelength in nanometers (nm), allowing researchers to choose the appropriate dye for their experimental needs. (Engel et al., 2014)

Alexa Fluor 488, the most popular dye in the series, is a chemically modified version of fluorescein. It maintains a similar emission spectrum to its original variant, but with improved performance in fluorescence imaging applications. Its more persistent and brighter fluorescence, photostability, and compatibility with most fluorescence detection instruments offers advantages for precise imaging. iFluor 488 can be used as an alternative dye to Alexa Fluor 488. (Lindhoud et al., 2012)

Two other popular dyes in the Alexa Fluor family are Alexa Fluor 568 and Alexa Fluor 594. These two dyes differ mainly in their excitation and emission wavelengths, with Alexa Fluor 594 emitting slightly further in the red spectrum. While both are versatile and compatible with a variety of biomolecules, the choice between them is based on experimental needs, such as the presence of other fluorescence dyes, desired level of specificity, and available detection equipment. Texas Red and iFluor 594 are alternative dyes to Alexa Fluor 594.

On the longer wavelength side, there is Alexa Fluor 750. Like Cy7, Alexa Fluor 750 is also a near-infrared fluorescent dye. However, Alexa Fluor 750 is more photostable, exhibits brighter fluorescence, and is less prone to quenching compared to Cy7. (Berlier et al., 2003)

As mentioned, one of the primary applications of Alexa Fluor dyes is to conjugate them to antibodies for bioassay detection. To prepare samples using Alexa-conjugated antibodies, samples must be incubated with the antibodies at room temperature or 4 °C as recommended, and stored in the dark to avoid photobleaching. The antibodies should be diluted in a blocking buffer to minimize non-specific binding and analyzed using an appropriate fluorescence detection method, such as a flow cytometer or fluorescence microscope.

Berlier, J. E., Rothe, A., Buller, G., Bradford, J., Gray, D. R., Filanoski, B. J., Telford, W. G., Yue, S., Liu, J., Cheung, C. Y., Chang, W., Hirsch, J. D., Beechem, J. M., Haugland, R. P., & Haugland, R. P. (2003). Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 51(12), 1699–1712. https://doi.org/10.1177/002215540305101214

Engel, U. (2014). Structured illumination superresolution imaging of the cytoskeleton. In Methods in Cell Biology (Vol. 123, pp. 315-333). Academic Press.

Lutea A.A. de Jong, ... Rainer Bischoff, in Journal of Chromatography B, 2005

Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, et al. Alexa Dyes, a Series of New Fluorescent Dyes that Yield Exceptionally Bright, Photostable Conjugates. Journal of Histochemistry & Cytochemistry. 1999;47(9):1179-1188. doi:10.1177/002215549904700910

Rasmussen, J. A. M., & Hermetter, A. (2008). Chemical synthesis of fluorescent glycero-and sphingolipids. Progress in lipid research, 47(6), 436-460.

Original created on October 29, 2024, last updated on October 29, 2024
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Alexa Fluor

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