For many years the technical support and the scientific staff at Molecular Probes 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.
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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.
- 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.
Are Alexa Fluor® Dyes Cell-Permeant?
Are Alexa Fluor® Dyes Compatible With Common Fixatives, Buffers, Permeabilizing Agents Like Formaldehyde, Formalin, Paraffin, Xylene, Triton® X-100, etc.?
Is Alexa Fluor® 405 the Best Dye for Imaging At 405 Nm?
What are the 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|
† 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?
What is the usable pH range of Alexa Fluor® 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.|
Alexa Fluor® 647 vs. Cy®5 Dye
All of our dyes have been designed to have no loss of brightness after conjugation. Alexa Fluor® 647 dye is an excellent spectral match for Cy®5 dye but has higher photostability and produces highly fluorescent conjugates that are 4 to 5 times brighter than conjugates of Cy®5 dye (Figures 5, 6).
|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 (Figures 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?
What is the Brightest, Most Photostable, and Best Alexa Fluor® Dye?
What Are the Two Best Alexa Fluor® Dyes?
Do I Need to Buy New Emission Filters to Spectrally Match the Alexa Fluor® Dyes?
Table 1. Emission filters for Alexa Fluor® dyes.
|Color||Invitrogen™ 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|
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Can I buy an Alexa Fluor® dye without a reactive group?
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Are the Alexa Fluor® azides from the Click-iT® EdU kits available separately?
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Choose the optimal Alexa Fluor® dye for your antibody or protein labeling application with a concise and informative set of criteria—all on your desktop.