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The Alexa Fluor Dye Series—Note 1.1

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 reagents.ref These dyes, without exception, produce the best and brightest conjugates we have ever tested. 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.
  • Instrument compatibility—Absorption spectra of the Alexa Fluor conjugates are matched to the principal output wavelengths of common excitation sources (Fluorescence excitation sources—Table 23.10).
  • Color selection—Alexa Fluor conjugates are available in several distinct fluorescent colors, ranging from blue to red to near-infrared (Figure 1, Table 1).
  • 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.

Conventional fluorophores and their conjugates can often be replaced with spectrally similar Alexa Fluor dyes and conjugates without affecting optical filter choices or other instrumentation considerations (Table 2). Each of the Alexa Fluor dyes are listed in Table 1 and described in detail in Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3 and in the accompanying Section 1.3 data table.




Figure 1. Emission spectra for the Alexa Fluor dye series.

Table 1. Spectral properties of Alexa Fluor dyes

Alexa Fluor DyeAbsorption
Max (nm)
Emission
Max (nm)
Emission Color *Extinction
Coefficient **
Alexa Fluor 350346442Blue 19,000
Alexa Fluor 405401421Blue 35,000
Alexa Fluor 430433541 Yellow-green 15,000
Alexa Fluor 488496519Green 73,000
Alexa Fluor 514
518
540Green80,000
Alexa Fluor 532532553Yellow 81,000
Alexa Fluor 546556573Orange 112,000
Alexa Fluor 555555565Orange 155,000
Alexa Fluor 568578603 Red-orange 88,000
Alexa Fluor 594590617Red 92,000
Alexa Fluor 610612628Red 144,000
Alexa Fluor 633632647Far-red 159,000
Alexa Fluor 635633647Far-red 140,000
Alexa Fluor 647650665Far-red 270,000
Alexa Fluor 660663690Near-IR *** 132,000
Alexa Fluor 680679702Near-IR *** 183,000
Alexa Fluor 700702723Near-IR *** 205,000
Alexa Fluor 750749775Near-IR *** 290,000
Alexa Fluor 790784814Near-IR *** 260,000
* Typical emission color seen through the eyepiece of a conventional fluorescence microscope with appropriate filters.
** Extinction coefficient of dye at emission maximum in cm-1M-1.
*** Human vision is insensitive to light beyond ~650 nm; it is not possible to view far-red– and near-IR–fluorescent dyes.
 

Table 2. Alexa Fluor dye alternatives to several common dyes

If you are using...Try this Alexa Fluor dye
Coumarin (AMCA)
Alexa Fluor 350
Cy2 or Fluorescein (FITC)
Alexa Fluor 488
Cy3 or Tetramethylrhodamine (TRITC)Alexa Fluor 555
Rhodamine RedAlexa Fluor 568
Texas RedAlexa Fluor 594
Cy5Alexa Fluor 647
Cy5.5
Alexa Fluor 680
Cy7Alexa Fluor 750