Alexa Fluor® dyes  

For many years our technical support and scientific staff have been asked questions around the properties, use, and specific applications for Alexa Fluor® dyes. Here is a comprehensive set of frequently asked questions and their answers as put together by the Molecular Probes® technical support staff for the benefit of the researcher.

Applications—Labeling Molecules

Do you have a protocol for conjugation of amine-reactive Alexa Fluor® dyes to proteins?

Yes, we have protocols for labeling your biomolecule with amine-reactive Alexa Fluor® dyes, as well as with the sulfhydryl-reactive forms.

What should I use to specifically label my antibody or protein?

Antibodies, proteins, and peptides have primary amines and sulfhydryl groups that are readily labeled.  Oligonucleotides are also often synthesized with either an amine or sulfhydryl group as a reactive "handle". Amine groups are the primary choice for labeling biomolecules, making proteins and antibodies among the easiest biomolecules to label.
Life Technologies has developed a wide selection of amine-reactive moieties on our superior Alexa Fluor® dyes. Thiol-reactive reagents provide a means to modify a protein at a defined site with high specificity. Alexa Fluor® maleimides are excellent reagents for thiol-selective modification, quantitation, and analysis. The preferred bioconjugate should be highly fluorescent (i.e., have a high degree of labeling (DoL)), yet retain key functional parameters such as solubility, target affinity, and enzymatic activity.

How can I label my antibody/protein/oligo with an Alexa Fluor® dye?

We provide a full tutorial and easy-to-use selection tools to help you choose the appropriate reagent or kit to enable labeling of target molecules.


Applications—Cell Analysis

At what concentration should an Alexa Fluor® IgG conjugate be used as a secondary antibody?

We recommend typical dilutions as follows:

  • Imaging applications—1:500 to 1:1000
  • Flow cytometry— 1:2000 or higher.

Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. for the fluorophore- and biotin-labeled antibodies, including the phycoerythrin-, allophycocyanin-, and tandem-labeled antibodies, a final concentration of 1–10 μg/mL should be satisfactory for most immunohistochemical applications. for flow cytometry applications, 0.06–1.0 μg per 1 million cells should yield satisfactory results.

Do you offer a specific staining procedure (fix/permeabilization/block) for using Alexa Fluor® conjugates in immunocytochemistry (ICC) and immunohistochemistry (IHC)?

Although staining protocols may vary with application, the Alexa Fluor® conjugates can be used in place of any other secondary IgG conjugates that you are already using. They do not require a modification of your staining procedure.

Are Alexa Fluor® dyes cell-permeant?

No, they are too highly charged.

Are Alexa Fluor® dyes compatible with common fixatives, buffers, permeabilizing agents like formaldehyde, formalin, paraffin, xylene, Triton® X-100, etc.?

Alexa Fluor® dyes are compatible with Triton®, formaldehyde, and formalin. Paraffin processing of tissues likely will extract dyes of almost any type, including Alexa Fluor® dyes, though they are fine to use after the processing, such as for immunohistochemistry (IHC) imaging.

Is Alexa Fluor® 405 the best dye for imaging at 405 nm?

The "best" dye really depends on the filter's emission specifications. Both Alexa Fluor® 405 dye and Alexa Fluor® 430 dye will efficiently excite at that wavelength.


Chemical Properties

How hydrophobic/hydrophilic are Alexa Fluor® dyes compared to other standard counterparts?

All of the Alexa Fluor® dyes have been engineered to be more hydrophilic than dyes such as fluorescein and Texas Red® dye. Alexa Fluor® 594 is the most hydrophobic of all of the Alexa Fluor® dyes.

What can quench Alexa Fluor® dyes?

Just like all dyes, Alexa Fluor® dyes are susceptible to typical quenchers such as chromogens (trypan blue), quenchers (QSY®), and other compounds such as 1, 4-diazabicyclooctane (DABCO) and N-propyl gallate (NPG), which, ironically, are also used as antifades.

What are the fluorescence quantum yields (QY) and lifetimes of the Alexa Fluor® dyes?

Table 2 shows fluorescence quantum yields (QY) and lifetimes of the Alexa Fluor® dyes.

Table 2. Fluorescence quantum yields (QY) and lifetimes (τ) of Alexa Fluor® dyes.

Alexa Fluor® Dye * Quantum yield (QY)† Lifetime (τ (ns))‡
Alexa Fluor® 488 0.92 4.1 §
Alexa Fluor® 532 0.61 2.5
Alexa Fluor® 546 0.79  4.1
Alexa Fluor® 555 0.10 0.3
Alexa Fluor® 568 0.69 3.6 §
Alexa Fluor® 594 0.66 3.9 §
Alexa Fluor® 647 0.33 1.0
Alexa Fluor® 660 0.37 1.2 **
Alexa Fluor® 680 0.36 1.2
Alexa Fluor® 700 0.25 1.0
Alexa Fluor® 750 0.12 1.0
* Measurements were made on free succinimidyl ester derivatives in aqueous solutions. † For Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, and Alexa Fluor® 647 dyes, QY measurements were made in PBS (50 mM potassium phosphate, 150 mM NaCl, pH 7.2) at 22°C relative to fluorescein in 0.01 M NaOH (QY = 0.92). For Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, and Alexa Fluor® 750 dyes, QY measurements were made in PBS at 22°C relative to Alexa Fluor® 647 succinimidyl ester in PBS (QY = 0.33). ‡ Except for the footnoted values, lifetime measurements were made in water at 22°C. Data provided by ISS Inc. (Champaign, IL). § Lifetime measurement was provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc. ** Lifetime measurement was made in pH 7.5 buffer at 20°C by Pierre-Alain Muller, Max Planck Institute for Biophysical Chemistry, Göttingen.

What is the pH stability of Alexa Fluor® dyes?

Most of the Alexa Fluor® dyes are stable between pH 4 and 10.

What is the usable pH range of Alexa Fluor® dyes?

For most of the Alexa Fluor® dyes, we recommend a pH range of 6.5 to 8.5.


Performance

How are the Alexa Fluor® dyes better in performance than their standard counterparts such as fluorescein (FITC), Cy®3, Cy®5, Texas Red®, and allophycocyanin (APC) dyes?

Alexa Fluor® dyes make brighter and more photostable conjugates than do fluorescein (FITC), Cy®3, Cy®5, Texas Red®, or allophycocyanin (APC) dyes. Alexa Fluor® 488 vs. Fluorescein (FITC) or Cy®2 Fluorescein and Cy®2 conjugates rapidly quench as more fluorophores are added. The Alexa Fluor® 488 dye allows more fluorophores to be attached to the conjugate before self-quenching becomes apparent, leading to significantly brighter conjugates (Figure 1). This increased brightness means that you can use less conjugate in your experiments, reducing background fluorescence and stretching your research dollar, and enhanced sensitivity enables detection of low-abundance targets.

 

 

Figure 1. Comparison of the relative fluorescence of Alexa Fluor® 488 dye and FITC. Goat anti–mouse IgG conjugate fluorescence was determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of a reference dye, and multiplying by the dye:protein labeling ratio.

Alexa Fluor® 555 vs. Cy®3 dye Alexa Fluor® 555 dye has higher photostability and quantum yield than tetramethylrhodamine isothiocyanate (TRITC) and rhodamine derivatives, which allow more time for observation and image capture. Alexa Fluor® 555 dye also has superior fluorescence output per protein or nucleic acid conjugate, surpassing that of any other spectrally similar fluorophore-labeled protein, including Cy®3–labeled proteins (Figures 2, 3). Other benefits include pH-insensitive fluorescence over a broad pH range, and solubility in water for easier labeling and detection.

 

Figure 2. Brightness comparison of Molecular Probes® Alexa Fluor® 555 goat anti–mouse IgG antibody vs. Cy®3 goat anti–mouse IgG antibody conjugates commercially available from several other companies. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended, and incubated with either the Alexa Fluor® 555 or Cy®3 goat anti–mouse IgG antibody at equal concentrations. Red blood cells were lysed, and the samples were analyzed with a flow cytometer equipped with a 488 nm argon-ion laser and a 585 ± 21 nm bandpass emission filter.

 

 

Figure 3. Comparison of the relative fluorescence of goat anti–rabbit IgG antibody conjugates of the Alexa Fluor® 555 and Cy®3 dyes at different dye:protein ratios in the conjugate.

Alexa Fluor® 594 vs. Texas Red® dye In almost all applications, the Alexa Fluor® 594 dye conjugates outperform the standard Texas Red® dye conjugates in terms of photostability and brightness (Figure 4).

 

 

Figure 4. Comparison of the relative fluorescence of Alexa Fluor® 594 and Texas Red®-X goat anti–mouse IgG antibody F (ab´)2 fragment conjugates at different dye:protein ratios.

 

 

Figure 5. Comparison of the brightness of Alexa Fluor® 647 and Cy®5 dye antibody conjugates.

 

 

Figure 6. Photobleaching resistance of the red-fluorescent Alexa Fluor® 647, Alexa Fluor® 633, PBXL-3, and Cy®5 dyes and the fluorescent protein allophycocyanin, as determined by laser-scanning cytometry.

Alexa Fluor® dyes have superior photostability In conclusion, the superior photostability of the Alexa Fluor® dyes allows more time for image observation and capture, thereby permitting greater sensitivity and simplifying the detection of low-abundance targets (Figure 7).

 

 

Figure 7. Photobleaching profiles of cells stained with Alexa Fluor® 488 dye or fluorescein. Alexa Fluor® 488 dye and fluorescein conjugates of goat anti–mouse IgG antibody F(ab´)2 fragment were used to detect HEp-2 cells probed with human antinuclear antibodies. Samples were continuously illuminated, and images were collected every 5 seconds with a cooled CCD camera. Normalized intensity data demonstrate the difference in photobleaching rates.

What antifade mountant do you recommend for cells stained with Alexa Fluor® conjugates?

For the best publication-quality imaging data, we recommend ProLong® Gold Antifade Reagent, which requires letting the sample cure at least overnight, which helps obtain crisper images. If there is a need to image samples without waiting overnight, we recommend SlowFade® Gold Antifade Reagent. For your convenience, these reagents come mixed with or without the nuclear stain 4', 6-diamidino-2-phenylindol (DAPI).

What is the brightest, most photostable, and best Alexa Fluor® dye?

All things being equal with the appropriate filters, Alexa Fluor® 488 is brighter and more photostable than other Alexa Fluor® dyes.

What are the two best Alexa Fluor® dyes?

If you have a typical fluorescence microscope with a BGR filter and no other constraints, then Alexa Fluor® 488 and Alexa Fluor® 594 are good choices, as the blue dyes generally have lower intensity.

Do I need to buy new emission filters to spectrally match the Alexa Fluor® dyes?

No, you do not have to buy special filters for Alexa Fluor® dyes, as these dyes have nearly identical spectral properties and generally better quantum yield compared to standard dyes. You can use the same filters that you use for the standard dyes (Table 1).

Table 1. Emission filters for Alexa Fluor® dyes.

Color Alexa Fluor® dyes Standard dyes
Green Alexa Fluor® 488 Fluorecein (FITC); Cy®2
Orange Alexa Fluor® 555 Rhodamine (TRITC); Cy®3
Red Alexa Fluor®594 Texas Red® Cy®3.5
NIR Alexa Fluor® 647 APC; Cy®5


Product Formulation/Availability

Are Alexa Fluor® reactive dye vials (as in the Protein Labeling Kits or Monoclonal Antibody Kits) also available as stand-alone products?

Yes, the reactive forms of these dyes are available as stand-alone products.

Can I buy an Alexa Fluor® dye without a reactive group?

No, we don't sell the dyes as just the carboxylic acid, but in many cases the hydrazide form is suitable for your research needs

Are the Alexa Fluor® azides from the Click-iT® EdU kits available separately?

Yes, both the azide- and alkyne-tagged versions of Alexa Fluor® dyes are available. Keep in mind that Click-iT® EdU kits use azide-tagged dyes.