Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3

Page Contents

• Overview of the Alexa Fluor Dyes
• Green-Fluorescent Alexa Fluor Dyes
• Yellow-to-Red–Fluorescent Alexa Fluor Dyes
• Far-Red– and Near-Infrared–Fluorescent Alexa Fluor Dyes
• Blue-Fluorescent Alexa Fluor Dyes
• Alexa Fluor Protein and Nucleic Acid Labeling Kits
• Alexa Fluor Tandem Conjugates and Other Bioconjugates
• Signal Amplification with Alexa Fluor Dyes
• Data Table
• Ordering Information

The Alexa Fluor dyes produce exceptionally bright and photostable conjugates (Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4, The Alexa Fluor Dye Series—Note 1.1). The Alexa Fluor dyes share several significant attributes, including:

• Strong absorption at wavelengths of maximal output of common excitation sources
• Bright and unusually photostable fluorescence of their bioconjugates
• Good water solubility, which makes the reactive dyes easy to conjugate and the conjugates resistant to precipitation and aggregation
• Insensitivity of their absorption and emission spectra to pH over a broad range
• Well-differentiated spectra, providing many options for multicolor detection and fluorescence resonance energy transfer (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2)
• High quantum yields and long fluorescence lifetimes (Fluorescence quantum yields (QY) and lifetimes (T) for Alexa Fluor dyes—Table 1.5)
• Extremely high FRET efficiency, with calculated R₀ values of up to 84 Å between pairs of Alexa Fluor dyes (R<0> values for some Alexa Fluor dyes—Table 1.6) and up to 77 Å between Alexa Fluor dyes and some nonfluorescent quenchers (R<0> values for QSY and dabcyl quenchers—Table 1.11)

Alexa Fluor dyes set new standards for fluorophores and the bioconjugates prepared from them. The absorption spectra (Figure 1.3.1, Figure 1.3.2, Figure 1.3.3) of these superior fluorescent dyes—Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750 and Alexa Fluor 790 dyes—span the visible and infrared spectrum (Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4) and match the principal output wavelengths of common excitation sources. Because there are so many different Alexa Fluor dyes, we have had to develop a systematic strategy for naming them. We identify these dyes with the trademark Alexa Fluor followed by the optimal excitation wavelength in nm; for example, Alexa Fluor 488 dye is optimally excited by the 488 nm spectral line of the argon-ion laser.

With spectra almost identical to those of fluorescein (Figure 1.3.4), but with far greater conjugate fluorescence and significantly better conjugate photostability, Alexa Fluor 488 dye is indisputably the best green-fluorescent reactive dye available. Spectra of Alexa Fluor 555 dye are an almost perfect match to those of Cy3 dye (Figure 1.3.9), but conjugates of Alexa Fluor 555 dye are more fluorescent (Figure 1.3.12) and more photostable (Figure 1.3.13) than those of Cy3 dye. Similarly, spectra of Alexa Fluor 647 conjugates substantially match those of the Cy5 dye (Figure 1.3.16) and Alexa Fluor 680 and Alexa Fluor 750 dyes exhibit spectral properties similar to those of Cy5.5 and Cy7 dyes, respectively (Figure 1.3.21, Figure 1.3.22). Tandem conjugates of the long-wavelength Alexa Fluor dyes with R-phycoerythrin or allophycocyanin (Phycobiliproteins—Section 6.4) further expand the utility of this dye series in multiplex flow cytometry applications by shifting the phycobiliprotein emission profile relative to the available detection channels (Figure 1.3.24, Figure 1.3.25).

Figure 1.3.1 Absorption spectra of our ultraviolet and blue light–absorbing Alexa Fluor dyes.

Figure 1.3.2 Absorption spectra of our green, yellow and red light–absorbing Alexa Fluor dyes.

Figure 1.3.3 Absorption spectra of our far-red and infrared light–absorbing Alexa Fluor dyes. Alexa Fluor 635 dye, available conjugated to antibodies, streptavidin and phalloidin, is not included here but its absorption spectrum is very similar to that of Alexa Fluor 633 dye.

Alexa Fluor 488 Dye: A Superior Fluorescein Substitute

Based on our testing, publications and results reported by customers, Alexa Fluor 488 dye is by far the best fluorescein (FITC or FAM) substitute available for most applications (The Alexa Fluor Dye Series—Note 1.1). It is probably the best dye available for single-molecule detection of bioconjugates, for fluorescence correlation spectroscopy (Fluorescence Correlation Spectroscopy (FCS)—Note 1.3) and for fluorescence polarization measurements (Fluorescence Polarization (FP)—Note 1.4). This green-fluorescent dye exhibits several unique features:

• Fluorescence spectra almost identical to those of fluorescein, with excitation/emission maxima of 495/519 nm (Figure 1.3.4) and a fluorescence lifetime of ~4.1 nanoseconds (Fluorescence quantum yields (QY) and lifetimes (T) for Alexa Fluor dyes—Table 1.5)
• Strong absorption, with an extinction coefficient greater than 65,000 cm^-1M^-1
• Much greater photostability than fluorescein (Figure 1.3.5), allowing more time for observation and image capture ()
• pH-insensitive fluorescence between pH 4 and 10 (Figure 1.3.6)
• Water solubility, with no organic co-solvents required in labeling reactions, suggesting that the succinimidyl ester of Alexa Fluor 488 carboxylic acid (A20000, A20100) may be the ideal reagent for labeling amines of exposed cell-surface proteins of live cells
• Superior fluorescence output per protein conjugate, surpassing that of any other spectrally similar fluorophore-labeled protein, including fluorescein conjugates (Figure 1.3.7) and Cy2 conjugates of antibodies (Figure 1.3.8)

The monosuccinimidyl ester of Alexa Fluor 488 carboxylic acid is a mixture of two isomers and is available in a 1 mg or 5 mg unit size (A20000, A20100). The isomerically pure 5-isomer of Alexa Fluor 488 dye is also available as the more hydrolytically stable tetrafluorophenyl (TFP) ester (A30005, A37563, A37570) and sulfodichlorophenol (SDP) ester (A30052).

TFP and SDP esters are an improvement over the succinimidyl ester (NHS ester or SE) chemistry typically used to attach fluorophores or haptens to the primary amines of biomolecules. All three reactive chemistries produce the same strong amide bond between the dye or hapten and the compound of interest (see reaction schemes in Introduction to Amine Modification—Section 1.1), but TFP and SDP esters are less susceptible to spontaneous hydrolysis during conjugation reactions. Both Alexa Fluor 488 carboxylic acid TFP ester and Alexa Fluor 488 SDP ester are stable for several hours at the basic pH typically used for reactions—far outlasting succinimidyl esters. Alexa Fluor 488 carboxylic acid TFP ester is the amine-reactive dye included in most of our labeling kits for proteins, nucleic acids and oligonucleotides (Kits for Labeling Proteins and Nucleic Acids—Section 1.2; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4), including the Alexa Fluor 488 Microscale Protein Labeling Kit (A30006), the Alexa Fluor 488 Antibody Labeling Kit (A20181), the Alexa Fluor 488 Protein Labeling Kit (A10235), the ARES Alexa Fluor 488 DNA Labeling Kit (A21665) and the Alexa Fluor 488 Oligonucleotide Amine Labeling Kit (A20191). Alexa Fluor 488 carboxylic acid SDP ester is the amine-reactive dye included in the APEX Alexa Fluor 488 Antibody Labeling Kit (A10468).

Figure 1.3.4 Absorption and fluorescence emission spectra of fluorescein goat anti–mouse IgG antibody (F2761) and Alexa Fluor 488 goat anti–mouse IgG antibody (A11001). The fluorescence intensity of the Alexa Fluor 488 conjugate was significantly higher than that of the fluorescein conjugate. The data are normalized to show the spectral similarity.

Figure 1.3.5 Photobleaching resistance of the green-fluorescent Alexa Fluor 488, Oregon Green 488 and fluorescein dyes, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 488 (S11223, S32354), Oregon Green 488 or fluorescein (S869) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). The cells were then fixed in 1% formaldehyde, washed and wet-mounted. After mounting, cells were scanned 10 times on a laser-scanning cytometer; laser power levels were 25 mW for the 488 nm spectral line of the argon-ion laser. Scan durations were approximately 5 minutes, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.

Figure 1.3.6 Comparison of pH-dependent fluorescence of the Oregon Green 488 (), carboxyfluorescein () and Alexa Fluor 488 () fluorophores. Fluorescence intensities were measured for equal concentrations of the three dyes using excitation/emission at 490/520 nm.

Figure 1.3.7 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates prepared from Alexa Fluor 488 dye and from fluorescein isothiocyanate (FITC). Conjugate fluorescence is 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.

Figure 1.3.8 Brightness comparison of Molecular Probes Alexa Fluor 488 goat anti–mouse IgG antibody with Cy2 goat anti–mouse IgG antibody from Jackson ImmunoResearch. 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 Alexa Fluor 488 or Cy2 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 525 ± 10 nm bandpass emission filter.

Alexa Fluor 514 Dye: A Perfect Match to the Argon-Ion Laser

Like our Alexa Fluor 488 dye, Alexa Fluor 514 dye (excitation/emission maxima ~518/540 nm) is superior to fluorescein in both brightness and photostability and can be detected with standard fluorescein, Oregon Green dye or Alexa Fluor 488 dye filter sets. However, Alexa Fluor 514 dye is spectrally distinguishable from Alexa Fluor 488 dye and other green fluorophores using spectral imaging instruments with linear-unmixing software. Alexa Fluor 514 dye is one the brightest and most photostable dyes available for excitation by the 514 nm spectral line of the argon-ion laser. Alexa Fluor 514 dye is available as a succinimidyl ester (A30002) and as antibody (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1) and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9) conjugates.

Alexa Fluor 430 Dye: Filling the Spectral Gap between Green and Yellow

Few reactive dyes that absorb between 400 nm and 450 nm have appreciable fluorescence beyond 500 nm in aqueous solution. Alexa Fluor 430 dye fills this spectral gap (Figure 1.34, ). Excitation near its absorption maximum at 431 nm is accompanied by an extremely large Stokes shift and strong yellow-green fluorescence (emission maximum ~541 nm). The coumarin-based amine-reactive succinimidyl ester of Alexa Fluor 430 carboxylic acid (A10169) is available, as well as Alexa Fluor 430 conjugates of secondary antibodies (A11063, A11064; Secondary Immunoreagents—Section 7.2) and streptavidin (S11237, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). Alexa Fluor 430 dye–labeled Fab fragments are provided in the Zenon Alexa Fluor 430 Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7).

As with the green-fluorescent Alexa Fluor 488 dye, the yellow-, orange- and red-fluorescent Alexa Fluor dyes exhibit several features that distinguish them from spectrally similar fluorophores:

• Strong absorption, with extinction coefficients greater than 80,000 cm^-1M^-1 for Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 594 dyes and greater than 130,000 cm^-1M^-1 for Alexa Fluor 555 and Alexa Fluor 610 dyes
• Fluorescence that is more photostable than that of other spectrally similar dyes, allowing more time for observation and image capture
• pH-insensitive fluorescence over a broad range
• Good water solubility, permitting labeling reactions to be performed without organic solvents
• Superior fluorescence output per protein or nucleic acid conjugate, surpassing that of any other spectrally similar fluorophore-labeled protein
• Long fluorescence lifetimes (approximately 4.1, 3.6 and 3.9 nanoseconds for Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 594 dyes, respectively (Fluorescence quantum yields (QY) and lifetimes (T) for Alexa Fluor dyes—Table 1.5))

Alexa Fluor 532 Dye: Optimal Dye for 532 nm Diode Lasers

The yellow-fluorescent Alexa Fluor 532 dye (excitation/emission maxima ~532/554 nm) absorbs in the wavelength range between the green-fluorescent Alexa Fluor 488 dye and orange-fluorescent Alexa Fluor 546 dye (Figure 1.3.2) and provides strong visible fluorescence that contrasts well with these dyes. Five of our Alexa Fluor dyes—Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 594 dyes—have been utilized for simultaneous seven-color fluorescence imaging in tissue samples. The use of Alexa Fluor 532 dye–labeled actin (instead of the equivalent tetramethylrhodamine conjugate) has been reported to be crucial for studying the polarity of individual filaments during the formation of actin bundles by total internal reflection fluorescence (TIRF) microscopy. The isomer-free, amine-reactive monosuccinimidyl ester of Alexa Fluor 532 dye is available in either a 1 mg or 5 mg unit size (A20001, A20101MP) and as a component of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4). The contents and utility of these protein and nucleic acid labeling kits are discussed in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Alexa Fluor 546 and Alexa Fluor 555 Dyes: Superior Alternatives to Cy3 and Tetramethylrhodamine

The orange-fluorescent Alexa Fluor 546 (excitation/emission maxima ~556/573 nm) and Alexa Fluor 555 (excitation/emission maxima ~555/565 nm) dyes have spectra that are similar to tetramethylrhodamine and the Cy3 dye. The spectra of Alexa Fluor 555 dye are an almost exact match to those of the Cy3 dye (Figure 1.3.9), and therefore optical filters designed for Cy3 dye also work with Alexa Fluor 555 dye. Conjugates of Alexa Fluor 546 and Alexa Fluor 555 dyes typically outperform tetramethylrhodamine (TRITC and TAMRA) and Cy3 conjugates (Figure 1.3.10, Figure 1.3.11), and Alexa Fluor 555 conjugates are more fluorescent at a higher degree of substitution (DOS) than are Cy3 conjugates (Figure 1.3.12). Alexa Fluor 555 dye is also more photostable than Cy3 dye (Figure 1.3.13), providing more time for image capture.

We have observed that, unlike most other Alexa Fluor dyes, antifade reagents provide little protective effect for conjugates of Alexa Fluor 546 dye; if photobleaching is a limitation, the spectrally similar Alexa Fluor 555 dye should be used in place of Alexa Fluor 546 dye. The isomeric mixture of the amine-reactive monosuccinimidyl ester of Alexa Fluor 546 dye (A20002, A20102) and the isomer-free monosuccinimidyl ester of Alexa Fluor 555 dye (A20009, A20109) are available in either a 1 mg or 5 mg unit size; Alexa Fluor 555 succinimidyl ester is offered in additional unit sizes (25 mg, A37564; three vials of 100 µg each, A37571). These reactive orange-fluorescent Alexa Fluor dyes are also available as components of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluo active esters and kits for labeling proteins and nucleic acids—Table 1.4). The contents and utility of these protein and nucleic acid labeling kits are discussed in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Figure 1.3.9 Comparison of the absorption and fluorescence emission spectra of Alexa Fluor 555 and Cy3 dyes. Spectra have been normalized to the same intensity for comparison purposes.

Figure 1.3.10 Fluorescence output from an Alexa Fluor 546 goat anti–mouse IgG antibody (dye:protein ratio = 5.7) and a commercially available Cy3 goat anti–mouse IgG antibody (dye:protein ratio = 3.8). Antibody concentrations were adjusted to give equal absorbance at the excitation wavelength (535 nm). The relative fluorescence quantum yield of Alexa Fluor 546 conjugates is higher than that of Cy3 conjugates, even at high dye:protein ratios that would typically result in self-quenching effects with most other protein-labeling dyes.

Figure 1.3.11 Brightness comparison of Alexa Fluor 555 goat anti–mouse IgG antibody with commercially available Cy3 goat anti–mouse IgG antibody conjugates. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells ere washed, resuspended and incubated with either Alexa Fluor 555 or Cy3 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 1.3.12 Comparison of the relative fluorescence of goat anti–rabbit IgG antibody conjugates of Alexa Fluor 555 and Cy3 dyes (prepared in our laboratories) at different dye:protein ratios in the conjugate.

Figure 1.3.13 Photobleaching profiles of Alexa Fluor 555 and Cy3 dyes were obtained by placing equal molar concentrations of the free dyes into capillary tubes; the samples were continuously illuminated and data points were collected every 5 seconds. Fluorescence has been normalized to the same initial intensity.

Alexa Fluor 568 Dye: A Perfect Match to 561 nm Diode Lasers

The red-orange–fluorescent Alexa Fluor 568 dye (excitation/emission maxima ~578/603 nm, ) is optimally excited by 561 nm diode lasers used in many confocal laser-scanning microscopes. Although Alexa Fluor 568 conjugates exhibit absorption and fluorescence emission maxima similar to those of Lissamine rhodamine B conjugates, they are considerably brighter. The isomeric mixture of the amine-reactive monosuccinimidyl ester of Alexa Fluor 568 dye is available in either a 1 mg or 5 mg unit size (A20003, A20103) and as a component of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4). The contents and utility of these protein and nucleic acid labeling kits are discussed in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Alexa Fluor 594 and Alexa Fluor 610 Dyes: Brighter Red-Fluorescent Dyes

The red-fluorescent Alexa Fluor 594 dye (excitation/emission maxima ~590/617 nm) has absorption and fluorescence emission maxima similar to those of Texas Red dye, making it particularly useful for multilabeling experiments in combination with green-fluorescent probes. Alexa Fluor 594 conjugates are brighter than similarly labeled Texas Red conjugates, can be labeled to a higher degree of substitution (DOS) (Figure 1.3.14), and are efficiently excited by 561 nm diode lasers and the 594 nm line of the orange He-Ne laser.

The bright and photostable Alexa Fluor 610 dye (excitation/emission maxima ~612/628 nm) emits an intense red fluorescence that is easily distinguished from green fluorescence and can be visualized with the same optics used for Texas Red and Alexa Fluor 594 dyes. Unlike the fluorescence of Alexa Fluor 633 dye and longer-wavelength fluorophores, Alexa Fluor 610 fluorescence can still be seen with the human eye.

The isomeric mixture of the amine-reactive monosuccinimidyl ester of Alexa Fluor 594 dye is available in units of 1 mg, 5 mg, 25 mg or three vials of 100 µg each (A20004, A20104, A37565, A37572), and the 6-isomer of the monosuccinimidyl ester of Alexa Fluor 610-X dye is available in a 1 mg unit size (A30050). These reactive red-fluorescent Alexa Fluor dyes are also available as components of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4); the contents and utility of these protein and nucleic acid labeling kits are discussed in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Figure 1.3.14 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.

One of our long-term goals has been to develop superior dyes that can be excited by long-wavelength excitation sources, including the red He-Ne laser (at 633 nm), krypton-ion laser (at 647 nm) and diode lasers. Long-wavelength excitation generally provides increased optical penetration of tissues and decreased autofluorescence background, both key enabling prerequisites for in vivo imaging applications. It has been particularly challenging to prepare long-wavelength reactive dyes whose fluorescence is not significantly quenched upon conjugation. Our far-red– and near-infrared–fluorescent Alexa Fluor dyes (Figure 1.3.3) meet our goals in several ways:

• Excellent spectral match to common long-wavelength excitation sources
• Very high extinction coefficients—typically >165,000 cm^-1M^-1 but up to 290,000 cm^-1M^-1 for Alexa Fluor 750 dye
• Spectra of Alexa Fluor 647, Alexa Fluor 680 and Alexa Fluor 750 conjugates that virtually match those of the Cy5 dye (Figure 1.3.16), Cy5.5 dye (Figure 1.3.21) and Cy7 dye (Figure 1.3.22), respectively, resulting in an optimal match to optical filters designed for these dyes
• Photostability of Alexa Fluor 633 and Alexa Fluor 647 conjugates that exceeds that of Cy5, allophycocyanin and PBXL-3 conjugates (Figure 1.3.15)
• Unusually low fluorescence quenching upon conjugation to proteins, even at relatively high degrees of substitution (Figure 1.3.17), resulting in protein conjugates that are typically at least three to four times brighter than those of Cy5, Cy5.5, Cy7 and similar dyes but that are, in some cases, as much as 40-fold brighter at equal antibody concentrations (Figure 1.3.17, Figure 1.3.18, Figure 1.3.19)
• Fluorescence of the nucleotide, oligonucleotide and nucleic acid conjugates of Alexa Fluor 647 dye that usually exceeds that of the Cy5 dye conjugates (Labeling Oligonucleotides and Nucleic Acids—Section 8.2)
• Unlike the Cy5 dye, very little change in absorption or fluorescence spectra when conjugated to most proteins, oligonucleotides and nucleic acids (Figure 1.3.20), thus yielding significantly greater total fluorescence at the same degrees of substitution (Figure 1.3.17, Figure 1.3.18, Figure 1.3.19)
• Reasonable water solubility of their succinimidyl esters, permitting conjugations to be done without addition of organic solvents if desired
• Chemistry that permits synthesis of pure, monofunctional reactive dyes, thus avoiding crosslinking reactions

The lifetimes and fluorescence quantum yields of far-red and near-infrared Alexa Fluor dyes are generally shorter and lower, respectively, than the their counterparts in the green-orange-red visible wavelength (488–594 nm) excitation range (Fluorescence quantum yields (QY) and lifetimes (τ) for Alexa Fluor dyes—Table 1.5). However, the lower quantum yields are compensated by larger extinction coefficients in terms of total fluorescence output. Fluorescence of these long-wavelength Alexa Fluor dyes is not visible to the human eye but is readily detected by most imaging systems. Pictures of these dyes throughout the Molecular Probes Handbook have been pseudocolored to represent the staining that is observed with sensitive detection equipment.

Alexa Fluor 633 and Alexa Fluor 635 Dyes: Optimal Excitation with the He-Ne Laser

These far-red–fluorescent Alexa Fluor dyes are important labels for fluorescence imaging because their spectra are beyond the range of most sample autofluorescence. With an excitation maximum of 633 nm and 635 nm, respectively, Alexa Fluor 633 and Alexa Fluor 635 dyes are a perfect match to the 633 nm spectral line of the He-Ne laser and the 635 nm spectral line of red diode lasers. Although their fluorescence is not visible to the human eye, Alexa Fluor 633 and Alexa Fluor 635 conjugates are bright and photostable (Figure 1.3.15), with peak emission at 647 nm.

An isomeric mixture of the amine-reactive succinimidyl ester of Alexa Fluor 633 dye is available as a stand-alone reagent in either a 1 mg or 5 mg unit size (A20005, A20105) and as a component of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4), which are described in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2. Alexa Fluor 635 dye, which is currently available as antibody (Summary of Molecular Probes secondary antibody conjugates—Table 7.1) and streptavidin (Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9) conjugates, typically produces brighter protein conjugates than does Alexa Fluor 633 dye because it is less susceptible to self-quenching.

Figure 1.3.15 Photobleaching resistance of the far-red–fluorescent Alexa Fluor 647, Alexa Fluor 633, PBXL-3 and Cy5 dyes and the allophycocyanin fluorescent protein, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 647 (S21374, S32357), Alexa Fluor 633 (S21375), PBXL-3, Cy5 or allophycocyanin (APC, S868) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). The cells were then fixed in 1% formaldehyde, washed and wet-mounted. After mounting, cells were scanned eight times on a laser-scanning cytometer; laser power levels were 18 mW for the 633 nm spectral line of the He-Ne laser. Scan durations were approximately 5 minutes, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.

Alexa Fluor 647 Dye: A Superior Alternative to Cy5 Dye

Spectra of Alexa Fluor 647 conjugates are virtually identical to those of the Cy5 dye (Figure 1.3.16), resulting in an optimal match to optical filters designed for that dye. Total fluorescence of Alexa Fluor 647 secondary antibody conjugates, however, is significantly higher that that of Cy5 conjugates commercially available from other suppliers (Figure 1.3.17, Figure 1.3.18, Figure 1.3.19). Also, unlike Cy5 dye, Alexa Fluor 647 dye has very little change in absorption or fluorescence spectra upon conjugation to most proteins and nucleic acids (Figure 1.3.20), thus yielding greater total fluorescence at the same degree of substitution. The spectral characteristics of thirteen different red-fluorescent fluorophores, including Alexa Fluor 647 and BODIPY 630/660 (BODIPY Dye Series—Section 1.4) dyes, have been evaluated in different surrounding media to assess the influence of polarity, viscosity and detergent concentration and to facilitate probe choice in fluorescence-based assays. Determinations of photobleaching quantum yields show that Alexa Fluor 647 dye is more photostable than Cy5. The monosuccinimidyl ester of Alexa Fluor 647 dye is available as a stand-alone reagent in units of 1 mg, 5 mg, 25 mg or three vials of 100 µg each (A20006, A20106, A37566, A37573) and as a component of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4), which are described in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Figure 1.3.16 Comparison of the fluorescence spectra of Alexa Fluor647 and Cy5 dyes. Spectra have been normalized to the same intensity for comparison purposes.

Figure 1.3.17 Comparison of the brightness of Alexa Fluor 647 and Cy5 dye antibody conjugates (prepared in our laboratories). More Alexa Fluor 647 dye molecules can be attached to proteins and nucleic acids without significant quenching, thus yielding conjugates that are much brighter than those possible using the Cy5 dye.

Figure 1.3.18 Flow cytometry was used to compare the brightness of Molecular Probes Alexa Fluor 647 goat anti–mouse IgG antibody (red, A21235) with commercially available Cy5 goat anti–mouse IgG antibody from Jackson ImmunoResearch Laboratories (green) and Amersham-Pharmacia Biotech (blue). 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 an Alexa Fluor 647 or Cy5 goat anti–mouse IgG secondary antibody at equal concentration. Red blood cells were lysed and the samples were analyzed on a flow cytometer equipped with a 633 nm He-Ne laser and a longpass emission filter (>650 nm).

Figure 1.3.19 Brightness comparison of Molecular Probes Alexa Fluor 647 goat anti–mouse IgG antibody with Cy5 goat anti–mouse IgG antibody conjugates commercially available from 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 Alexa Fluor 647 or Cy5 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 633 nm He-Ne laser and a longpass emission filter (>650 nm).

Figure 1.3.20 The absorption spectra of Cy5 dye conjugates of both proteins and nucleic acids show an additional peak at about 600 nm when compared to the spectrum of the free dye. However, the light absorbed by Cy5 conjugates at this wavelength does not result in fluorescence. Alexa Fluor 647 protein conjugates do not exhibit this spectral anomaly. Spectra have been normalized to the same peak intensity for comparison purposes.

Alexa Fluor 660 Dye: A Match for the Krypton-Ion Laser

Alexa Fluor 660 is optimally excited by the 647 nm spectral line of the krypton-ion laser and well excited by the 633 nm spectral line of the He-Ne laser. Protein conjugates of Alexa Fluor 660 dye produce bright near-infrared fluorescence, with a peak at 690 nm. This long-wavelength emission is well separated from that of other fluorophores, including Alexa Fluor 546 and Cy3 dyes and phycoerythrin conjugates. Alexa Fluor 660 dye is also the dye of choice as a second label for use with allophycocyanin (APC) conjugates in flow cytometry applications. The monosuccinimidyl ester of Alexa Fluor 660 dye is available as a 1 mg stand-alone reagent (A20007) and as a component of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4), which are described in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Alexa Fluor 680 Dye: An Alternative to the Cy5.5 Dye

With a peak excitation at 679 nm and maximum emission at 702 nm, Alexa Fluor 680 dye is spectrally similar to Cy5.5 dye (Figure 1.26). Fluorescence emission of Alexa Fluor 680 dye is well separated from that of other commonly used red fluorophores, such as the tetramethylrhodamine, Texas Red, R-phycoerythrin, Alexa Fluor 594 and Alexa Fluor 647 dyes, making it ideal for three- and four-color labeling. The monosuccinimidyl ester of Alexa Fluor 680 dye is available as a stand-alone reagent in units of 1 mg, 5 mg, 25 mg or three vials of 100 µg each (A20008, A20108, A37567, A37574) and as a component of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4), which are described in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Figure 1.3.21 Comparison of the fluorescence spectra of the unconjugated Alexa Fluor 680 and Cy5.5 dyes. Spectra have been normalized to the same intensity for comparison purposes.

Alexa Fluor 700, Alexa Fluor 750 and Alexa Fluor 790 Dyes: Our Longest-Wavelength Dyes

With an absorption maximum at 702 nm, Alexa Fluor 700 dye can be excited with a xenon-arc lamp, far-red diode lasers or dye-pumped lasers operating in the 675–700 nm range. Alexa Fluor 700 dye provides near-infrared fluorescence emission, with a peak at 723 nm. Alexa Fluor 750 dye exhibits fluorescence spectra that are very similar to those of Cy7 dye (Figure 1.27). Its fluorescence emission maximum at 775 nm is well separated from commonly used far-red fluorophores such as Alexa Fluor 647, Alexa Fluor 660 or allophycocyanin (APC), facilitating multicolor analysis. With a peak excitation at 749 nm, conjugates of Alexa Fluor 750 dye are well excited by a xenon-arc lamp or dye-pumped lasers operating in the 720–750 nm range. The monosuccinimidyl esters of Alexa Fluor 700 (A20010, A20110) and Alexa Fluor 750 (A20011, A20111) dyes are available as stand-alone reagents in either a 1 mg or 5 mg unit size; Alexa Fluor 750 dye is offered in additional unit sizes (25 mg, A37568; three vials of 100 µg each, A37575). These reactive Alexa Fluor dyes are also available as components of several labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4), which are described in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Alexa Fluor 790 dye is the longest-wavelength Alexa Fluor dye available. With excitation/emission maxima of 784/814 nm, Alexa Fluor 790 dye has spectral properties similar to those of indocyanine green (ICG) and IRDye 800 dyes (LI-COR Biosciences). This fluorophore is especially useful for researchers who require an amine-reactive, near-infrared label for small animal in vivo imaging (SAIVI) applications, as well as for multicolor analysis with Alexa Fluor 680 dye and the LI-COR Odyssey infrared imaging system. The monosuccinimidyl ester of Alexa Fluor 790 dye is supplied in units of 100 µg (A30051), which is enough to label ~1 mg of IgG antibody, and 10 mg (A37569). This reactive long-wavelength Alexa Fluor dye is also a component of the Alexa Fluor 790 Antibody Labeling Kit (A20189, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4), which is described in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2.

Figure 1.3.22 Comparison of the fluorescence emission spectra of Alexa Fluor 750 and Cy7 dyes. Spectra have been normalized to the same intensity for comparison purposes.

Because their structures are closely related to those of the coumarins and pyrenes, the blue-fluorescent Alexa Fluor 350 and Alexa Fluor 405 dyes, as well as the yellow-green–fluorescent Alexa Fluor 430 dye described above, are also included in Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7. We summarize their properties here to complete our discussion of the Alexa Fluor dye series.

Alexa Fluor 350 Dye: Brighter Blue Fluorescence

The blue-fluorescent Alexa Fluor 350 carboxylic acid succinimidyl ester (A10168) is a sulfonated coumarin derivative that is more water soluble than either AMCA succinimidyl ester or AMCA-X succinimidyl ester (Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7) and yields protein conjugates that are typically 50% more fluorescent than those prepared from its nonsulfonated analog (Figure 1.3.23). Alexa Fluor 350 protein conjugates are optimally excited at 346 nm (Figure 1.3.1) and exhibit bright blue fluorescence at wavelengths slightly shorter than AMCA or AMCA-X conjugates (442 nm versus 448 nm), which reduces the dye's spectral overlap with the emission of fluorescein. We also prepare Alexa Fluor 350 conjugates of secondary antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1) and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9), as well as several Alexa Fluor 350 protein labeling kits, which are described in detail in Kits for Labeling Proteins and Nucleic Acids—Section 1.2 (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4).

Figure 1.3.23 Comparison of the relative fluorescence of 7-amino-4-methylcoumarin-3-acetic acid (AMCA) streptavidin () and Alexa Fluor 350 streptavidin, a sulfonated AMCA derivative (S11249, ). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein.

Alexa Fluor 405 Dye: Near-Perfect Match to the Violet Diode Laser

With excitation/emission maxima of 402/421 nm (Figure 1.34), the blue-fluorescent Alexa Fluor 405 dye is well matched to the 405 nm spectral line of violet diode lasers now widely implemented in fluorescence microscopy and flow cytometry. Alexa Fluor 405 succinimidyl ester is an amine-reactive derivative of our Cascade Blue dye, which was previously available in amine-reactive form only as its acetyl azide (C2284, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7). Not only is it offered at higher purity than the alternative Cascade Blue acetyl azide, but Alexa Fluor 405 succinimidyl ester also contains a 4-piperidinecarboxylic acid spacer that separates the fluorophore from its reactive moiety. This spacer enhances the reactivity of the succinimidyl ester and minimizes any interactions between the fluorophore and the biomolecule to which it is conjugated.

As with Cascade Blue acetyl azide, Alexa Fluor 405 dye shows minimal spectral overlap with green fluorophores, making it ideal for multicolor applications. Alexa Fluor 405 dye is available as a succinimidyl ester (A30000, A30100) and a cadaverine derivative (A30675, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4), as well as conjugated to secondary antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1) and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9).

Alexa Fluor 405 dye is also recognized by our anti–Alexa Fluor 405/Cascade Blue dye antibody (A5760, Anti-Dye and Anti-Hapten Antibodies—Section 7.4). In addition, Alexa Fluor 405 dye–labeled Fab fragments are provided in the Zenon Alexa Fluor 405 Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7).

Alexa Fluor Labeling Kits

As described above, all of our Alexa Fluor dyes are available as amine-reactive succinimidyl esters (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids—Table 1.4), and the Alexa Fluor 488 dye is additionally available as its single-isomer, hydrolysis-resistant tetrafluorophenyl (TFP) ester (A30005). Most of these amine-reactive Alexa Fluor dyes are also offered as components of several protein and nucleic acid labeling kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2), which are described thoroughly in Kits for Labeling Proteins and Nucleic Acids—Section 1.2 and include:

• APEX Antibody Labeling Kits
• Microscale Protein Labeling Kits
• Alexa Fluor Antibody Labeling Kits
• SAIVI Rapid Antibody Labeling Kits
• Alexa Fluor Protein Labeling Kits
• Zenon Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7)
• ARES DNA Labeling Kits (Labeling Oligonucleotides and Nucleic Acids—Section 8.2)
• FISH Tag DNA Kits and FISH Tag RNA Kits (Labeling Oligonucleotides and Nucleic Acids—Section 8.2)
• Alexa Fluor Oligonucleotide Amine Labeling Kits (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Molecular Probes nucleic acid labeling kits—Table 8.6)
• ULYSIS Nucleic Acid Labeling Kits (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Molecular Probes nucleic acid labeling kits—Table 8.6)

The purity of the Alexa Fluor carboxylic acid succinimidyl esters dyes when prepared and packaged in a 5 mg unit size is usually >80–95% by HPLC. However, Alexa Fluor dyes tenaciously bind water, and packaging of these products in smaller unit sizes—such as the multiple vials used in the Alexa Fluor labeling kits and the 1 mg stand-alone reagents—may result in some loss of reactivity. Our specifications for Alexa Fluor carboxylic acid succinimidyl esters provided as kit components or as stand-alone reagents require that the product has ≥50% reactivity after packaging.

Alexa Fluor Decapacks for Labeling Amine-Modified DNA or RNA

For labeling amine-modified DNA or RNA probes in microarray-based experiments, we offer the Alexa Fluor 488 reactive dye decapack (A32750), Alexa Fluor 555 reactive dye decapack (A32756) and Alexa Fluor 647 reactive dye decapack (A32757), which provide the corresponding Alexa Fluor succinimidyl ester conveniently packaged in 10 single-use vials. These specially packaged amine-reactive Alexa Fluor dyes can be used in conjunction with the aminoallyl dUTP (Labeling Oligonucleotides and Nucleic Acids—Section 8.2) nucleotide or with commercially available aminoallyl nucleotide–based nucleic acid labeling kits.

Each single-use vial contains sufficient Alexa Fluor succinimidyl ester to optimally label the amount of cDNA produced from reverse transcription of either 20 µg of total RNA or 1–5 µg of poly(A)+ RNA, in the presence of aminoallyl dUTP. For added convenience, we offer a combination set of the Alexa Fluor 555 and Alexa Fluor 647 reactive dye decapacks (A32755) that contains 10 vials of each succinimidyl ester and is sufficient for 10 two-color labeling reactions. The Alexa Fluor 555/Alexa Fluor 647 dye pair has been shown to display higher signal correlation coefficients than the Cy3/Cy5 dye pair in two-color DNA microarray assays.

Other Reactive Alexa Fluor Derivatives

Several Alexa Fluor dyes are also available as thiol-reactive maleimides (Thiol-Reactive Probes Excited with Visible Light—Section 2.2, Molecular Probes thiol-reactive dyes excited with visible light—Table 2.1), as alkynes and azides for bioorthogonal "click" reactions (Click Chemistry—Section 3.1) and as aldehyde- and ketone-reactive hydrazides and hydroxylamines (Reagents for Modifying Aldehydes and Ketones—Section 3.3, Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2). The Alexa Fluor hydrazides and hydroxylamines are also important probes for intracellular tracing (Polar Tracers—Section 14.3). Although some of the Alexa Fluor dyes are mixtures of two isomers, all the reactive Alexa Fluor dyes have only a single reactive substituent linked to each fluorophore.

Alexa Fluor Dye–Phycobiliprotein Tandem Conjugates

We have conjugated R-phycoerythrin with Alexa Fluor 610, Alexa Fluor 647 or Alexa Fluor 680 dye—and in turn conjugated these fluorescent proteins to antibodies or streptavidin, yielding tandem conjugates that permit simultaneous multicolor labeling and detection of multiple targets using a single excitation source (the 488 nm spectral line of the argon-ion laser) and monitoring emission at 628 nm, 668 nm or 702 nm, respectively (Phycobiliproteins—Section 6.4, Figure 1.3.24). Additionally, we have conjugated allophycocyanin to Alexa Fluor 680, Alexa Fluor 700 or Alexa Fluor 750 dyes to create tandem conjugates for multicolor measurements using excitation sources in the 633 to 650 nm range (Figure 1.3.25). Zenon Antibody Labeling Kits for the rapid and quantitative labeling of antibodies with the phycobiliprotein tandem dyes are also available (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7).

Figure 1.3.24 Normalized fluorescence emission spectra of 1) Alexa Fluor 488 goat anti–mouse IgG antibody (A11001), 2) R-phycoerythrin goat anti–mouse IgG antibody (P852), 3) Alexa Fluor 610–R-phycoerythrin goat anti–mouse IgG antibody (A20980), 4) Alexa Fluor 647–R-phycoerythrin goat anti–mouse IgG antibody (A20990) and 5) Alexa Fluor 680–R-phycoerythrin goat anti–mouse IgG antibody. The tandem conjugates permit simultaneous multicolor labeling and detection of up to five targets with excitation by a single excitation source—the 488 nm spectral line of the argon-ion laser.<

Figure 1.3.25Normalized fluorescence emission spectra of 1) allophycocyanin goat anti–mouse IgG antibody (A865), 2) Alexa Fluor 680–allophycocyanin goat anti–mouse IgG antibody and 3) Alexa Fluor 750–allophycocyanin goat anti–mouse IgG antibody (A21006). The tandem conjugates permit simultaneous multicolor labeling and detection of up to three targets with excitation by a single excitation source—the 633 nm spectral line of the He-Ne laser.

Other Alexa Fluor Bioconjugates

For immunofluorescence, receptor labeling, nucleic acid synthesis, cell tracing and many other applications, we offer a wide variety of Alexa Fluor conjugates, including labeled antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1), streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9) and many other proteins, ligands and nucleotides.

Tyramide Signal Amplification

Tyramide signal amplification (TSA) technology, which was developed by NEN (now a part of PerkinElmer Corporation) and licensed for in-cell and in-tissue applications, permits significant amplification of cellular targets by a horseradish peroxidase (HRP)–mediated scheme (Figure 1.3.26). We have introduced several TSA Kits (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2, Tyramide Signal Amplification (TSA) Kits—Table 6.1), including kits that utilize one of the following Alexa Fluor tyramides:

• Alexa Fluor 350 tyramide ()
• Alexa Fluor 488 tyramide ()
• Alexa Fluor 546 tyramide
• Alexa Fluor 555 tyramide
• Alexa Fluor 568 tyramide ()
• Alexa Fluor 594 tyramide
• Alexa Fluor 647 tyramide () 

The HRP-catalyzed immobilization of a fluorescent tyramide can yield far greater total fluorescence than would ever be possible with direct labeling of the target, enabling detection of very low-abundance receptors (Figure 1.3.27). Furthermore, TSA can be used in either live- or fixed-cell preparations, and the increased sensitivity of this signal amplification method often permits use of greatly decreased quantities of antibodies or nucleic acid probes. Our complete selection of TSA Kits are listed in Tyramide Signal Amplification (TSA) Kits—Table 6.1 and are extensively discussed in TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2.

Figure 1.3.26 Schematic representation of TSA detection methods applied to immunolabeling of an antigen. The antigen is detected by a primary antibody, followed by a horseradish peroxidase–labeled secondary antibody in conjunction with a dye-labeled (or hapten-labeled) tyramide, resulting in localized deposition of the activated tyramide derivative (Stage 1). Further dye deposition, and therefore higher levels of signal amplification, can be generated by detecting dye deposited in Stage 1 with a horseradish peroxidase–labeled anti-dye antibody in conjunction with a dye-labeled tyramide (Stage 2).

Figure 1.3.27 Detection of epidermal growth factor (EGF) receptors directly or with signal amplification. Cells expressing high (A431 cells, panel A) and low (NIH 3T3 cells, panel B) levels of EGF receptors were either directly labeled with the preformed Alexa Fluor 488 complex of biotinylated epidermal growth factor (E13345, blue) or indirectly labeled with biotinylated EGF (E3477) followed by either Alexa Fluor 488 streptavidin (S11223, green) or HRP-conjugated streptavidin and Alexa Fluor 488 tyramide (B40953, purple).

Antibody-Based Signal Amplification Kits

Although the direct fluorescence signal of Alexa Fluor conjugates tends to be significantly greater than that of other dyes with comparable spectra, we have also developed two kits that take further advantage of the superior brightness and photostability of Alexa Fluor 488 dye– and Alexa Fluor 594 dye–labeled reagents. These Alexa Fluor Signal Amplification Kits are designed to substantially increase the signals obtained by immunofluorescence techniques (Figure 1.3.28), thus permitting detection of low-abundance targets. The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes provides methods and reagents for two-stage amplification of fluorescein (FITC)–labeled primary antibodies and also overwrites the fluorescein fluorescence signal with that of the brighter and more photostable Alexa Fluor 488 dye (Figure 1.3.29). The Alexa Fluor 488 Signal Amplification Kit for Mouse Antibodies (A11054) provides two-stage amplification of immunocytochemical labeling generated by mouse primary antibodies. The similar Alexa Fluor 568 Signal Amplification Kit for Mouse Antibodies (A11066) provide ultrasensitive immunofluorescent detection at longer wavelengths. For additional details about these kits, see Secondary Immunoreagents—Section 7.2.

Figure 1.3.28 An example of flow cytometry results obtained using the Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes. Human T cell leukemia cells (Jurkat) were stained with fluorescein (FITC) mouse anti-CD4 antibody and, as indicated, with Alexa Fluor 488 rabbit anti–fluorescein/Oregon Green antibody (A11090) and Alexa Fluor 488 goat anti–rabbit IgG antibody (A11008). The fluorescence values of the negative controls, in which the FITC anti-CD4 antibody was omitted, are shown (black) together with the fluorescence values of the experimental samples (green). The fluorescence values represent the average signals from the population of cells analyzed.

Figure 1.3.29 Demonstration of the amplification obtained with the Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes. Bovine pulmonary artery endothelial cells were labeled with anti–α-tubulin antibody (A11126) in combination with fluorescein goat anti–mouse IgG antibody (F2761) (left panel). The center panel shows the cells after treatment with Alexa Fluor 488 rabbit anti–fluorescein/Oregon Green antibody (A11090), and the right panel shows the cells after additional labeling with Alexa Fluor 488 goat anti–rabbit IgG antibody (A11008). The images were acquired using identical exposure times, and a bandpass filter set appropriate for fluorescein.

Alexa Fluor Conjugates of Anti–Fluorescein/Oregon Green Antibody

Alexa Fluor 488 dye–labeled rabbit anti–fluorescein/Oregon Green antibody (A11090, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) can be used to enhance the green-fluorescent signal of the fluorescein (or Oregon Green) hapten without changing its fluorescence color. Thus, this conjugate allows researchers to take advantage of the superior photostability of Alexa Fluor 488 dye, while utilizing existing fluorescein- or Oregon Green dye–labeled probes and fluorescein-compatible optics. Alexa Fluor 594 dye–labeled rabbit anti–fluorescein/Oregon Green antibody (A11091) can be used to convert the green fluorescence of fluorescein or Oregon Green conjugates into exceptionally photostable red fluorescence (), and to amplify the signal from fluorescein and Oregon Green conjugates by as much as 100-fold (Figure 1.3.30).

Figure 1.3.30 Color-shifting using a labeled anti–fluorescein/Oregon Green antibody. Jurkat cells were first stained with a primary mouse anti–human CD3 antibody, followed by fluorescein goat anti–mouse IgG antibody (F2761), with the resultant fluorescence detected in the R-phycoerythrin (red-orange fluorescence) channel of a flow cytometer (blue curve). The weak signal was then shifted to better suit the R-phycoerythrin channel by the addition of an R-phycoerythrin conjugate of anti–fluorescein/Oregon Green antibody (A21250). The resulting signal intensity is approximately two orders of magnitude greater (red curve) than the direct fluorescence from the first staining step (blue curve).

Antibodies to Alexa Fluor 488 and Alexa Fluor 405 Dyes

We offer a rabbit polyclonal antibody to Alexa Fluor 488 dye (A11094, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) that quenches the dye's fluorescence and can be used in various signal amplification schemes, including further amplification of the signal from Alexa Fluor conjugates of proteins or nucleic acids or potentially from Alexa Fluor 488 tyramide (B40953, TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2).

As expected, the rabbit polyclonal antibody to the Cascade Blue dye (A5760, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) strongly interacts with Alexa Fluor 405 dye, making it useful for various fluorescence quenching and amplification schemes. Our Zenon Rabbit IgG Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7) can also be used to prepare fluorescent dye–, biotin- or enzyme-labeled complexes of these rabbit IgG antibodies for use in various detection and amplification schemes.

For a detailed explanation of column headings, see Definitions of Data Table Contents