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

Figure 1.34 Absorption spectra of our short-wavelength light–absorbing Alexa Fluor dyes.



Figure 1.17 Absorption spectra of our intermediate-wavelength light–absorbing Alexa Fluor dyes.



Figure 1.24 Absorption spectra of our long-wavelength light–absorbing Alexa Fluor dyes. The 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 the Alexa Fluor 633 dye.



Figure 1.8 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 1.18 Comparison of the absorption and fluorescence emission spectra of the Alexa Fluor 555 and Cy3 dyes. Spectra have been normalized to the same intensity for comparison purposes.



Figure 1.29 Comparison of the relative fluorescence of goat anti–rabbit IgG antibody conjugates of the Alexa Fluor 555 and Cy3 dyes (prepared by Molecular Probes, Inc.) at different dye:protein ratios in the conjugate.



Figure 1.20 Photobleaching profiles of the 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 five seconds. Fluorescence has been normalized to the same initial intensity.



Figure 1.25 Comparison of the fluorescence spectra of the Alexa Fluor 647 and Cy5 dyes. Spectra have been normalized to the same intensity for comparison purposes.



Figure 1.26 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.



Figure 1.27 Comparison of the fluorescence emission spectra of the Alexa Fluor 750 and Cy7 dyes. Spectra have been normalized to the same intensity for comparison purposes.



Figure 6.34 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 (A20983). 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 6.37 Normalized fluorescence emission spectra of 1) allophycocyanin goat anti–mouse IgG antibody (A865), 2) Alexa Fluor 680–allophycocyanin goat anti–mouse IgG antibody (A21000) 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.

Our exclusive Zenon Antibody Labeling Kits, which are available for most of the Alexa Fluor dyes (Active esters and kits for labeling proteins and nucleic acids - Table 1.2, Molecular Probes' Zenon Labeling Kits - Table 7.14), make it possible to rapidly and quantitatively label antibodies from a purified antibody fraction or from a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant (Figure 7.56). The Zenon Antibody Labeling Kits and the Zenon technology are described in detail in Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3.

The Alexa Fluor series of dyes shares several significant attributes, including:

• High absorbance at wavelengths of maximal output of common excitation sources
• Bright and unusually photostable fluorescence of their bioconjugates
• Good water solubility of the reactive dyes for ease of conjugation and resistance of the conjugates to precipitation and aggregation
• Insensitivity of their 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 R_o 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 (Molecular Probes' amine-reactive dyes - Table 1.1)

Figure 7.56 Labeling scheme utilized in the Zenon Antibody Labeling Kits. An unlabeled IgG antibody is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled, Fc-specific anti-IgG Fab fragment (panel A). This labeled Fab fragment binds to the Fc portion of the IgG antibody (panel B). Excess Fab fragment is then neutralized by the addition of a nonspecific IgG (panel C), preventing crosslabeling by the Fab fragment in experiments where primary antibodies of the same type are present. Note that the Fab fragment used for labeling need not be coupled to a fluorophore, but could instead be coupled to an enzyme (such as HRP) or to biotin.

Alexa Fluor 488 Dye

Based on our testing, publications and results reported by customers, the Alexa Fluor 488 dye is by far the best fluorescein (FITC or FAM) substitute available for most applications (Customer Testimonials for the Alexa Fluor Dyes - Note 1.3 ). It is probably the best dye available for single-molecule detection of bioconjugates, for fluorescence correlation spectroscopy (Fluorescence Correlation Spectroscopy (FCS) - Note 1.4 ) and for fluorescence polarization measurements (Fluorescence Polarization (FP) - Note 1.5). 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 1.8) 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 1.9), allowing more time for observation and image capture (, )
• pH-insensitive fluorescence between pH 4 and 10 (Figure 1.12)
• 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 1.13) and Cy2 conjugates of antibodies (Figure 1.14)
• Utility as a fluorescence anisotropy probe for measuring protein–protein interactions (Fluorescence Polarization (FP) - Note 1.5)

Figure 1.9 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), Oregon Green 488 (S6368) 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% paraformaldehyde, 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 five minutes apiece, 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 1.12 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 1.13 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates prepared from the 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 1.14 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 concentration. 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.

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; Alexa Fluor(R) Succinimidyl Esters). The isomerically pure 5-isomer of the Alexa Fluor 488 dye is also available as an amine-reactive tetrafluorophenyl (TFP) ester (A30005, ). TFP 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. Both reactive chemistries produce the same strong amide bond between the dye or hapten and the compound of interest (Figure 1.2), but TFP esters are less susceptible to spontaneous hydrolysis during conjugation reactions (Figure 1.16). The Alexa Fluor 488 carboxylic acid TFP ester is stable for several hours at the basic pH typically used for reactions — far outlasting succinimidyl esters. The amine-reactive Alexa Fluor 488 succinimidyl ester is a component of several 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); the Alexa Fluor 488 carboxylic acid TFP ester is the amine-reactive dye included in the Alexa Fluor 488 Monoclonal Antibody Labeling Kit (A20181), the Alexa Fluor 488 Protein Labeling Kit (A10235) and the Alexa Fluor 488 Microscale Protein Labeling Kit (A30006).

Figure 1.2 Reaction of a primary amine with a succinimidyl ester or a tetrafluorophenyl (TFP) ester.

Figure 1.16 Stability of the tetrafluorophenyl (TFP) and succinimidyl (NHS) esters at basic pH (8.0–9.0).

For labeling amine-modified DNA or RNA probes in microarray-based experiments,, we offer the Alexa Fluor 488 reactive dye decapack (A32750), which provides our outstanding Alexa Fluor 488 succinimidyl ester conveniently packaged in 10 single-use vials. This specially packaged amine-reactive dye can be used in conjunction with our aminohexylacrylamido-dUTP (aha-dUTP, A32760; Labeling Oligonucleotides and Nucleic Acids - Section 8.2), aminoallyl dUTP or aminoallyl UTP (A21664, A21663; Labeling Oligonucleotides and Nucleic Acids - Section 8.2) nucleotides or with commercially available aminoallyl nucleotide–based nucleic acid labeling kits. The Alexa Fluor 488 succinimidyl ester produces high-efficiency labeling of aminoallyl-modified DNA or RNA — up to one dye every 12 bases. With excitation/emission maxima of 495/519 nm, the Alexa Fluor 488 succinimidyl ester matches one of the most popular wavelength channels used to scan microarrays. 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. We also offer the Alexa Fluor 555, Alexa Fluor 594 and Alexa Fluor 647 reactive dye decapacks (A32756, A32751, A32757; see below), and, for added convenience, 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.

Alexa Fluor 500 and Alexa Fluor 514 Dyes

Sophisticated detection systems demand highly specialized Alexa Fluor dyes. Instruments such as the Zeiss META system, with the capacity to differentiate between fluorescence emission maxima <5 nm apart, greatly expand the palette of fluorescent colors available for multicolor labeling experiments. To keep up with the capabilities and demands of these advancing technologies, Molecular Probes has developed the Alexa Fluor 500 and Alexa Fluor 514 dyes, with visually similar but spectrally distinct emission profiles (Figure 1.17, , ). Like our Alexa Fluor 488 dye, these two green-fluorescent Alexa Fluor dyes are superior to fluorescein in both brightness and photostability, and they can be detected with standard fluorescein, Oregon Green dye or Alexa Fluor 488 dye filter sets (Optical Filters for Fluorescence Microscopy - Section 23.5, Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11). However, the Alexa Fluor 500 and Alexa Fluor 514 dyes are specifically designed to be detected simultaneously with other green fluorophores, despite their spectral overlap. Though they appear similar in color by eye, the Alexa Fluor 500 dye can be optically separated from the Alexa Fluor 514 dye using the Zeiss META system or similar spectral imaging instruments with linear-unmixing software. Similarly, the fluorescent signal from the Alexa Fluor 514 dye can be resolved from both the Alexa Fluor 488 and the Alexa Fluor 500 fluorescence emissions. Additionally, the 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. Both the Alexa Fluor 500 and the Alexa Fluor 514 dyes are available as succinimidyl esters (A30001, A30002) and as antibody (Secondary Immunoreagents - Section 7.2, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1) and streptavidin conjugates (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23).

 

Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 610 Dyes

These yellow- to orange- to red-fluorescent Alexa Fluor dyes (Figure 1.17) provide strong visible fluorescence that contrasts well with the green fluorescence of the Alexa Fluor 488 dye; consequently, they are frequently used in combination with green-fluorescent dyes. Five of our Alexa Fluor dyes have been utilized for simultaneous seven-color fluorescence imaging in tissue samples. The Alexa Fluor 532 dye () is readily excited by the frequency-doubled output of the Nd:YAG laser. Both the Alexa Fluor 546 and Alexa Fluor 555 dyes have spectra that are similar to tetramethylrhodamine and the Cy3 dye; the spectra of the Alexa Fluor 555 dye are an almost exact match to those of the Cy3 dye (Figure 1.18). We have observed that, unlike most other Alexa Fluor dyes, antifade reagents provide little protective effect for conjugates of the Alexa Fluor 546 dye; the spectrally similar Alexa Fluor 555 dye is a good substitute for the Alexa Fluor 546 dye in many applications. The Alexa Fluor 568 () and Alexa Fluor 594 dyes have absorption and fluorescence emission maxima similar to the Lissamine rhodamine B and Texas Red dyes, respectively. The Alexa Fluor 610 dye emits an intense red fluorescence that, unlike the Alexa Fluor 633 dye and longer-wavelength fluorophores, can still be seen with the human eye. Each of these yellow-, orange- and red-fluorescent Alexa Fluor dyes exhibits several features that distinguish them from spectrally similar fluorophores:

• Excitation/emission maxima of ~531/554 nm for the Alexa Fluor 532 dye (), ~556/573 nm for the Alexa Fluor 546 dye (), ~555/565 nm for the Alexa Fluor 555 dye (), ~578/603 nm for the Alexa Fluor 568 dye (), ~590/617 nm for the Alexa Fluor 594 dye () and ~612/628 nm for the Alexa Fluor 610 dye (), with fluorescence lifetimes for the Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 594 dyes of approximately 4.1, 3.6 and 3.9 nanoseconds, respectively (Fluorescence quantum yields (QY) and lifetimes (T) for Alexa Fluor dyes - Table 1.5)
• Strong absorption, with extinction coefficients greater than 80,000 cm^-1M^-1 for the Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 594 dyes and greater than 130,000 cm^-1M^-1 for the 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 (Figure 1.20)
• pH-insensitive fluorescence over a broad range
• Water solubility, therefore 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 (Figure 1.21), including Cy3 dye–labeled proteins (Figure 1.22, Figure 1.23)

Figure 1.21 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 1.22 Brightness comparison of Molecular Probes' Alexa Fluor 555 goat anti–mouse IgG antibody with Cy3 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 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 1.23 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.

Isomeric mixtures of the amine-reactive monosuccinimidyl esters of the Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 594 dyes and the isomer-free monosuccinimidyl esters of the Alexa Fluor 532, Alexa Fluor 555 and Alexa Fluor 610 dyes are available as separate reagents in either a 1 mg or 5 mg unit size (Alexa Fluor(R) Succinimidyl Esters) or 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.

For labeling amine-modified DNA or RNA probes in microarray-based experiments, we offer the Alexa Fluor 555 and the Alexa Fluor 594 reactive dye decapacks (A32756, A32751), which provide our outstanding Alexa Fluor 555 and Alexa Fluor 594 succinimidyl esters, respectively, conveniently packaged in 10 single-use vials. This specially packaged amine-reactive dye can be used in conjunction with our aminohexylacrylamido-dUTP (aha-dUTP, A32760; Labeling Oligonucleotides and Nucleic Acids - Section 8.2), aminoallyl dUTP or aminoallyl UTP (A21664, A21663; Labeling Oligonucleotides and Nucleic Acids - Section 8.2) nucleotides or with commercially available aminoallyl nucleotide–based nucleic acid labeling kits. The monoreactive, single-isomer Alexa Fluor 555 succinimidyl ester produces high-efficiency labeling of aminoallyl-modified DNA or RNA — up to one dye every 12 bases. With excitation/emission maxima of 555/565 nm, the Alexa Fluor 555 succinimidyl ester matches one of the most popular wavelength channels used to scan microarrays. Conjugates of the Alexa Fluor 594 succinimidyl ester (excitation/emission maxima ~590/617 nm) exhibit very little spectral overlap with green-fluorescent conjugates and are efficiently excited by the 568 nm line of the Ar–Kr laser and by the 594 nm line of the orange He–Ne laser. Furthermore, the Alexa Fluor 555/Alexa Fluor 647 dye pair have been shown to display higher signal correlation coefficients than the Cy3/Cy5 dye pair in two-color DNA microarray assays. 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. We also offer the Alexa Fluor 488 reactive dye decapack (A32750, see above) and the Alexa Fluor 647 reactive dye decapack (A32757, see below), and, for added convenience, 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.

Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 Dyes

A long-term goal at Molecular Probes 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 laser diodes. It has particularly been a challenge to prepare reactive dyes whose fluorescence is not significantly quenched on conjugation. The Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes (Figure 1.24) meet our goals in several ways:

• An excellent spectral match to common long-wavelength excitation sources, with very high extinction coefficients — typically >165,000 cm^-1M^-1 but up to >230,000 cm^-1M^-1 for the Alexa Fluor 750 dye
• Spectra of the Alexa Fluor 647, Alexa Fluor 680 and Alexa Fluor 750 conjugates that virtually match those of the Cy5 dye (Figure 1.25), Cy5.5 dye (Figure 1.26) and Cy7 dye (Figure 1.27), respectively, resulting in an optimal match to optical filters designed for these dyes (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11)
• Photostability of the Alexa Fluor 633 and Alexa Fluor 647 conjugates that exceeds that of Cy5, allophycocyanin and PBXL-3 conjugates (Figure 1.28)
• Unusually low fluorescence quenching upon conjugation to proteins, even at relatively high degrees of substitution (Figure 1.29, Figure 1.13, Figure 1.30), 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 1.26, Figure 1.30, Figure 1.31, Figure 1.32)
• Fluorescence of the nucleotide, oligonucleotide and nucleic acid conjugates of the Alexa Fluor 647 dye that usually exceeds that of the Cy5 dye conjugates (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Detecting Nucleic Acid Hybridization - Section 8.5)
• Unlike the Cy5 dye, very little change in absorbance or fluorescence spectra when conjugated to most proteins, oligonucleotides and nucleic acids (Figure 1.33), thus yielding significantly greater total fluorescence at the same degrees of substitution (Figure 1.30, Figure 1.31, Figure 1.32)
• 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, singly reactive dyes, thus avoiding crosslinking reactions

Figure 1.28 Photobleaching resistance of the 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), 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% paraformaldehyde, 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 five minutes apiece, 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 1.30 Comparison of the brightness of Alexa Fluor 647 and Cy5 dye antibody conjugates (prepared by Molecular Probes, Inc.). 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 1.31 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 1.32 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 an equal concentration. 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 1.33 The absorption spectra of the 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, light absorbed by the Cy5 dye conjugates at this wavelength does not result in fluorescence. Alexa Fluor 647 dye conjugates of proteins do not exhibit this spectral anomaly. Spectra have been normalized to the same peak intensity for comparison purposes.

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 this Handbook have been pseudocolored to represent the staining that is observed with sensitive detection equipment.

An isomeric mixture of the amine-reactive succinimidyl ester of the Alexa Fluor 633 dye and the isomer-free monosuccinimidyl esters of the Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes are available as stand-alone reagents in either a 1 mg or 5 mg unit size (Active esters and kits for labeling proteins and nucleic acids - Table 1.2, Alexa Fluor(R) Succinimidyl Esters), and in most cases, as components of kits that permit facile labeling of proteins, oligonucleotides and nucleic acids (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). These kits and their contents are described in detail in Kits for Labeling Proteins and Nucleic Acids - Section 1.2. The Alexa Fluor 635 dye, which is currently only available as antibody (Summary of Molecular Probes' secondary antibody conjugates - Table 7.1, ) and streptavidin (Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23) conjugates, typically produces brighter protein conjugates than does the Alexa Fluor 633 dye because the absorption spectrum of the Alexa Fluor 635 dye does not split into two peaks upon protein conjugation, as do the absorption spectra of the Alexa Fluor 633, Cy5 and tetramethylrhodamine dyes (Figure 1.71). The spectral characteristics of thirteen different red-fluorescent fluorophores, including the 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.

Figure 1.71 Effect of protein conjugation on the absorption spectrum of tetramethylrhodamine. The absorption spectrum of tetramethylrhodamine conjugated to goat anti–mouse IgG antibody (TMR-GAM, T2762) shows an additional peak at about 520 nm when compared with the spectrum of the same concentration of the free dye (TMR). Partial unfolding of the protein in the presence of 4.8 M guanidine hydrochloride (TMR-GAM + GuHCl) results in a spectrum more similar to that of the free dye.

For labeling amine-modified DNA or RNA probes in microarray-based experiments,, we offer the Alexa Fluor 647 reactive dye decapack (A32757), which provides our outstanding Alexa Fluor 647 succinimidyl ester conveniently packaged in 10 single-use vials. This specially packaged amine-reactive dye can be used in conjunction with our aminohexylacrylamido-dUTP (aha-dUTP, A32760; Labeling Oligonucleotides and Nucleic Acids - Section 8.2), aminoallyl dUTP or aminoallyl UTP (A21664, A21663; Labeling Oligonucleotides and Nucleic Acids - Section 8.2) nucleotides or with commercially available aminoallyl nucleotide–based nucleic acid labeling kits. The Alexa Fluor 647 succinimidyl ester produces high-efficiency labeling of aminoallyl-modified DNA or RNA — up to one dye every 12 bases. With excitation/emission maxima of 650/668 nm, the Alexa Fluor 647 succinimidyl ester matches one of the most popular wavelength channels used to scan microarrays. Furthermore, the Alexa Fluor 555/Alexa Fluor 647 dye pair have been shown to display higher signal correlation coefficients than the Cy3/Cy5 dye pair in two-color DNA microarray assays. 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. We also offer the Alexa Fluor 488, Alexa Fluor 555 and Alexa Fluor 594 reactive dye decapacks (A32750, A32756, A32751; see above), and, for added convenience, 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.

Alexa Fluor 350 Dye

The sulfonated coumarin derivative, Alexa Fluor 350 carboxylic acid succinimidyl ester (), is more water soluble than either AMCA succinimidyl ester or AMCA-X succinimidyl ester (A6118, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores - Section 1.7) and yields protein conjugates that are more fluorescent than those prepared from its nonsulfonated analog (Figure 7.31). Alexa Fluor 350 protein conjugates are optimally excited at 346 nm (Figure 1.34, ) 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.

Figure 7.31 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

With excitation/emission maxima of 402/421 nm (Figure 1.34, ), our Alexa Fluor 405 dye is a near-perfect match to the 405 nm spectral line of the blue diode laser recently developed for fluorescence microscopy and flow cytometry. The 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 is Cascade Blue acetyl azide, but the 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 conjugates of Cascade Blue acetyl azide, the Alexa Fluor 405 conjugates show minimal spectral overlap with green fluorophores, making them ideal for multicolor applications. Moreover, with its longer-wavelength excitation maximum, the Alexa Fluor 405 dye is potentially brighter than UV light–excitable blue fluorophores, whose signal is often obscured by autofluorescence. The Alexa Fluor 405 dye is available as a succinimidyl ester (A30000, A30100), a maleimide (A30458, Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3), a thiol-reactive mercurial (Hg-Link Alexa Fluor 405 phenylmercury, H30461; Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3) and a cadaverine (A30675, Derivatization Reagents for Carboxylic Acids and Glutamine - Section 3.3), 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' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23). The 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 tyramide is used in the Tyramide Signal Amplification (TSA) Kits (Tyramide Signal Amplification (TSA) Technology - Section 6.2, Tyramide Signal Amplification (TSA) Kits - Table 6.1), and 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, Molecular Probes' Zenon Labeling Kits - Table 7.14).

Alexa Fluor 430 Dye

Few reactive dyes that absorb between 400 nm and 450 nm have appreciable fluorescence beyond 500 nm in aqueous solution. Our Alexa Fluor 430 dye fills this spectral gap (Figure 1.34, , ). Excitation near its absorption maximum at 431 nm is accompanied by strong yellow-green fluorescence, with an emission maximum at 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, Molecular Probes' Zenon Labeling Kits - Table 7.14).

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, Alexa Fluor(R) Succinimidyl Esters), and the Alexa Fluor 488 dye is additionally available as its single-isomer, hydrolysis-resistant tetrafluorophenyl (TFP) ester (A30005). Most of the 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) that are principally discussed in Kits for Labeling Proteins and Nucleic Acids - Section 1.2, including:

• Easy-to-Use Protein Labeling Kits (Kits for Labeling Proteins and Nucleic Acids - Section 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids - Table 1.4)
• Monoclonal Antibody Labeling Kits (Kits for Labeling Proteins and Nucleic Acids - Section 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids - Table 1.4)
• Zenon Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3; Active esters and kits for labeling proteins and nucleic acids - Table 1.2, Molecular Probes' Zenon Labeling Kits - Table 7.14) for the easiest and fastest method of preparing labeled antibody complexes, particularly on a microgram or submicrogram scale
• ARES DNA Labeling Kits (Kits for Labeling Proteins and Nucleic Acids - Section 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids - Table 1.4; Labeling Oligonucleotides and Nucleic Acids - Section 8.2)
• Alexa Fluor Oligonucleotide Amine Labeling Kits (Kits for Labeling Proteins and Nucleic Acids - Section 1.2, Alexa Fluor active esters and kits for labeling proteins and nucleic acids - Table 1.4; Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Oligonucleotide Amine Labeling Kits - Table 8.10)
• ULYSIS Nucleic Acid Labeling Kits (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Spectral characteristics of the fluorescent dyes available in Molecular Probes' ULYSIS Nucleic Acid Labeling Kits - Table 8.8), which utilize Alexa Fluor conjugates of a guanosine-reactive platinum compound for labeling of intact nucleic acids

These kits and their components are described in detail in the sections and tables indicated above. In addition, we offer several ChromaTide UTP, ChromaTide dUTP, aha-dUTP and ChromaTide OBEA-dCTP nucleotides (Characteristics of ChromaTide UTP nucleotides - Table 8.6, Characteristics of ChromaTide dUTP, ChromaTide OBEA-dCTP, aha-dUTP and aha-dCTP labeled nucleotides - Table 8.7) that include our Alexa Fluor dyes for enzyme-catalyzed incorporation into nucleic acids. The ChromaTide and aha-dUTP nucleotides are described in Labeling Oligonucleotides and Nucleic Acids - Section 8.2.

Purity of the Alexa Fluor carboxylic acid succinimidyl esters dyes when prepared and when packaged in a 5 mg unit size (Active esters and kits for labeling proteins and nucleic acids - Table 1.2) is usually >80–95% by HPLC. However, Alexa Fluor dyes tenaciously bind water, and packaging of these products in smaller unit sizes — the 1 mg stand-alone reagents and the multiple vials used in all kits — may result in some loss of reactivity. The Alexa Fluor 488 tetrafluorophenyl (TFP) ester (A30005) has somewhat better resistance to water and may be the preferred amine-reactive form reactive of this exceptional reagent. Our specifications for stand-alone Alexa Fluor carboxylic acid succinimidyl esters that are sold in a 1 mg size or as a component of a labeling kit require the product to have reactivity >=50% after packaging. As part of our quality control protocol, we test the suitability of the reactive Alexa Fluor reagents in the 1 mg unit size and in all of our Alexa Fluor protein and nucleic acid labeling kits after packaging; however, we recommend that all of the Alexa Fluor carboxylic acid succinimidyl esters (Alexa Fluor(R) Succinimidyl Esters) and Alexa Fluor protein and nucleic acid labeling kits be used soon after receipt.

Several Alexa Fluor dyes are also available as thiol-reactive maleimides and mercurials (Thiol-Reactive Probes Excited with Visible Light - Section 2.2, Thiol-reactive dyes excited with visible light - Table 2.1) and as aldehyde- and ketone-reactive hydrazides and hydroxylamines (Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2, Molecular Probes' hydrazine, hydroxylamine and amine derivatives - Table 3.1). The Alexa Fluor hydrazides and hydroxylamines are also important probes for intracellular tracing (Polar Tracers - Section 14.3; , Figure 14.23). Although some of the Alexa Fluor dyes are mixtures of two isomers, all the reactive Alexa Fluor dyes contain only a single reactive moiety.

The Alexa Fluor fluorophores, reactive dyes, conjugates and their applications are the subject of several Patents and patent applications filed by Molecular Probes, Inc., and are offered for research purposes only. Molecular Probes welcomes inquiries about Licensing these products and technology for resale or other commercial uses. Custom conjugations of the Alexa Fluor fluorophores are also available. Please contact our Custom and Bulk Sales Department.

Figure 14.23 Confocal image stack of a 10,000 MW Calcium Green dextran–labeled (C3713, Fluorescent Ca{2+} Indicator Conjugates - Section 19.4) climbing fiber in a sagittal cerebellar slice, showing incoming axon and terminal arborization (in yellow). The Purkinje cell innervated by this climbing fiber was labeled with Alexa Fluor 568 hydrazide (A10437, A10441) via a patch pipette and visually identified using bright-field microscopy. Image contributed by Anatol Kreitzer, Department of Neurobiology, Harvard Medical School.

Alexa Fluor Bioconjugates

For immunofluorescence, receptor labeling, nucleic acid synthesis, cell tracing and many other applications, we offer Alexa Fluor dyes in a wide variety of bioconjugates, including those of:

• Tandem conjugates of R-phycoerythrin (R-PE) and allophycocyanin (APC) for multicolor applications that employ a single laser as an excitation source (Phycobiliproteins - Section 6.4; Figure 6.34, Figure 6.37)
• Zenon labeling reagents (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3; Active esters and kits for labeling proteins and nucleic acids - Table 1.2, Molecular Probes' Zenon Labeling Kits - Table 7.14), for quick and convenient labeling of antibodies for multicolor applications
• Secondary antibodies (Secondary Immunoreagents - Section 7.2, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1)
• Protein A and protein G (Secondary Immunoreagents - Section 7.2, Protein A and protein G conjugates - Table 7.13)
• FluoroNanogold antibody and streptavidin conjugates (Secondary Immunoreagents - Section 7.2, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6)
• Anti-fluorescein/Oregon Green antibody for simultaneously amplifying and photostabilizing the signal of fluorescein- or Oregon Green dye–conjugated probes (A11090, Anti-Dye and Anti-Hapten Antibodies - Section 7.4) and for changing the green fluorescence of these probes to red fluorescence (A11091, A21250; Anti-Dye and Anti-Hapten Antibodies - Section 7.4; , Figure 7.70)
• Anti-biotin and anti–dinitrophenyl-KLH antibodies (Anti-Dye and Anti-Hapten Antibodies - Section 7.4)
• Primary antibodies, including the CD3, CD4 and CD8 antibodies (Primary Antibodies for Diverse Applications - Section 7.5)
• Anti–green-fluorescent protein antibody (anti-GFP; A21311, A21312; Primary Antibodies for Diverse Applications - Section 7.5)
• An antibody to the epitope tag hemagglutinin (anti-HA; A21287, A21288; Primary Antibodies for Diverse Applications - Section 7.5)
• Anti–glutathione S-transferase antibody (A11131, Primary Antibodies for Diverse Applications - Section 7.5)
• Avidin and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23)
• Lectins (Lectins and Other Carbohydrate-Binding Proteins - Section 7.7, Molecular Probes' selection of lectin conjugates - Table 7.24)
• UTP, dUTP, aha-dUTP and OBEA-dCTP nucleotides (Labeling Oligonucleotides and Nucleic Acids - Section 8.2) for enzyme-mediated incorporation into nucleic acids
• Panomer 9 random oligodeoxynucleotide and oligodeoxythymidine-18 (dT₁₈) conjugates (Detecting Nucleic Acid Hybridization - Section 8.5, Spectral characteristics of labeled oligonucleotides - Table 8.18)
• Phalloidin, F-actin and DNase I (Probes for Actin - Section 11.1, Spectral characteristics of our F-actin-selective probes - Table 11.1)
• An antibody to glial fibrillary acidic protein (anti-GFAP, Probes for Tubulin and Other Cytoskeletal Proteins - Section 11.2)
• Anti–OxPhos Complex IV antibody (anti–complex I of cytochrome oxidase, Probes for Mitochondria - Section 12.2)
• Dextrans (Fluorescent and Biotinylated Dextrans - Section 14.5)
• Bovine serum albumin, parvalbumin, soybean trypsin inhibitor, α-crystallin and cholera toxin subunit B (Protein Conjugates - Section 14.7)
• Anti-bromodeoxyuridine antibody and kits for following cell proliferation and apoptosis (Assays for Cell Enumeration, Cell Proliferation and Cell Cycle - Section 15.4, Assays for Apoptosis - Section 15.5)
• Annexin V (Assays for Apoptosis - Section 15.5)
• Fibrinogen and methotrexate (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione - Section 15.6)
• Lipopolysaccharides (Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1, Fluorescent lipopolysaccharide conjugates - Table 16.1)
• Acetylated low-density lipoprotein (Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1)
• Escherichia coli, Staphylococcus aureus and zymosan A BioParticles, epidermal growth factor, histones and transferrin (Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1; Transferrin conjugates - Table 16.2, Molecular Probes' selection of BioParticles fluorescent bacteria and yeast - Table 16.3)
• Angiotensin II, neuromedin C and substance P (Probes for Neurotransmitter Receptors - Section 16.2)
• α-Bungarotoxin (Probes for Neurotransmitter Receptors - Section 16.2, Labeled and unlabeled alpha-bungarotoxins - Table 16.4)
• Apamin (Probes for Ion Channels and Carriers - Section 16.3)
• Calmodulin (Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins - Section 17.3)

Figure 7.70 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).

Alexa Fluor Tandem Conjugates of Phycobiliproteins

We have conjugated R-phycoerythrin with an Alexa Fluor 610 dye and with our Alexa Fluor 647 and Alexa Fluor 680 dyes — and in turn conjugated these fluorescent proteins to antibodies or streptavidin, yielding tandem conjugates that permit simultaneous multicolor labeling and detection of multiple targets with excitation by a single excitation source — the 488 nm spectral line of the argon-ion laser (Phycobiliproteins - Section 6.4, Figure 6.34). Additionally, our Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 tandem conjugates of allophycocyanin can be combined with allophycocyanin or Alexa Fluor 647 bioconjugates for multicolor measurements using excitation by the lasers that emit at 633 to 650 nm (Figure 6.37). Zenon Antibody Labeling Kits for the rapid and quantitative labeling of antibodies with the tandem phycobiliprotein dyes are also available (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14).

DyeMer Bifluorophores

Our DyeMer 488/605, DyeMer 488/615 and DyeMer 488/630 conjugates of secondary antibodies (Secondary Immunoreagents - Section 7.2, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1) and of streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23) are optimized for use in flow cytometry applications. The red-orange–fluorescent DyeMer 488/605, red-fluorescent DyeMer 488/615 and far-red–fluorescent DyeMer 488/630 conjugates are each labeled with a unique bifluorophore comprising two covalently linked fluorophores that act as a donor–acceptor pair for fluorescence resonance energy transfer (FRET). When the green-fluorescent donor dye is excited with the 488 nm spectral line of the argon-ion laser, efficient energy transfer produces fluorescence of the long-wavelength acceptor dye, which emits at 611, 617 or 630 nm (, , ). Any fluorescence from the donor dye due to incomplete FRET can easily be compensated for by setting up compensation circuits to remove unwanted signals. Although their total fluorescence is not as intense as that of the phycobiliprotein tandem conjugates, the DyeMer conjugates exhibit minimal lot-to-lot variation and less interference at the antigen- or biotin-binding site due to the relatively small size of the DyeMer bifluorophores. Moreover, their fluorescence can be excited either at 488 nm or at their longer-wavelength absorption maximum. Because there is some green fluorescence emitted from the donor dye, the DyeMer conjugates were not developed for imaging applications. By carefully choosing bandpass filters that block this green fluorescence or by using a green-fluorescent label for the most abundant target to keep exposure times short, these DyeMer conjugates can be successfully applied to multicolor fluorescence microscopy experiments.

Tyramide Signal Amplification

Tyramide signal amplification (TSA) technology, which was developed by NEN (now a part of PerkinElmer Corporation) and licensed to Molecular Probes for in-cell and in-tissue applications, permits significant amplification of cellular targets by a horseradish peroxidase (HRP)–mediated scheme (Figure 6.5). Molecular Probes has introduced several TSA Kits (Tyramide Signal Amplification (TSA) Technology - Section 6.2, Tyramide Signal Amplification (TSA) Kits - Table 6.1), including kits that utilize Alexa Fluor 350 tyramide (), Alexa Fluor 405 tyramide, Alexa Fluor 488 tyramide (, ), Alexa Fluor 546 tyramide, Alexa Fluor 555 tyramide, Alexa Fluor 568 tyramide (), Alexa Fluor 594 tyramide or Alexa Fluor 647 tyramide () as the amplification reagent. 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 6.10) — and can be used in either live- or fixed-cell preparations. TSA also permits use of greatly decreased quantities of precious antibodies or nucleic acid probes. Our TSA Kits are listed in Tyramide Signal Amplification (TSA) Kits - Table 6.1 and are extensively discussed in Tyramide Signal Amplification (TSA) Technology - Section 6.2.

Figure 6.5 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 6.10 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 (purple), components of our TSA Kit #22 (T20932).

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 7.47), thus permitting detection of low-abundance targets. The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes (A11053) dramatically enhances the fluorescence and photostability of virtually any fluoresceinated probe (Figure 7.46). The Alexa Fluor 488 Signal Amplification Kit for Mouse Antibodies (A11054) can be used to sensitively detect mouse primary antibodies. The similar Alexa Fluor 568 and Alexa Fluor 594 Signal Amplification Kits for Mouse Antibodies (A11066, A11067) provide ultrasensitive immunofluorescent detection at longer wavelengths. For additional details about these kits, see Secondary Immunoreagents - Section 7.2 and our product literature (Alexa Fluor(R) 568 Signal-Amplification Kit for Mouse Antibodies, Alexa Fluor(R) 594 Signal-Amplification Kit for Mouse Antibodies).

Figure 7.47 An example of flow cytometry results obtained using the Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes (A11053). 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 7.46 Demonstration of the amplification obtained with the Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes (A11053). 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 show the cells after additional labeling with the 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

Our 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 the Alexa Fluor 488 dye, while utilizing existing fluorescein- or Oregon Green dye–labeled probes and fluorescein-compatible optics (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11). The 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 7.70).

Antibodies to the Alexa Fluor 488 and Alexa Fluor 405 Dyes

We offer a rabbit polyclonal antibody to the 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, potentially including further amplification of the signal from the TSA Kits that contain Alexa Fluor 488 tyramide (T20912, T20922, T20932; Tyramide Signal Amplification (TSA) Technology - Section 6.2) or from Alexa Fluor conjugates of proteins or nucleic acids. As expected, the rabbit polyclonal antibody to the Cascade Blue dye that we developed (A5760, Anti-Dye and Anti-Hapten Antibodies - Section 7.4) strongly interacts with the 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, Molecular Probes' Zenon Labeling Kits - Table 7.14) 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.