A Wide Variety of Protein Conjugates - Section 7.1

Antibody and Avidin Conjugates

The quality of a conjugate depends to a large degree on the quality of the protein from which it is made, as well as on the spectral properties of the fluorophore, the dye-to-protein ratio (DOS) and the methods used for the conjugate's purification. Molecular Probes uses the highest-quality proteins in its conjugates. In addition, our dyes and conjugation methods yield conjugates that are typically brighter than other commercially available conjugates (Figure 7.36, Figure 7.37, Figure 7.38), yet have low background and often have better spectral resolution. We also take exceptional care to remove unconjugated dye from the conjugate during purification. Moreover, all of our primary and secondary antibody conjugates and our avidin, streptavidin, NeutrAvidin and CaptAvidin biotin-binding protein conjugates are tested on cell samples to ensure low nonspecific binding and high specific staining. Summary of Molecular Probes' secondary antibody conjugates - Table 7.1, Molecular Probes' goat anti-mouse isotype-specific antibodies - Table 7.5 and Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23 list our current offerings of fluorescent secondary immunoreagents and avidins. We also offer biotin, DSB-X biotin (a readily reversible version of biotin; Figure 4.1, Figure 7.100, ) and enzyme conjugates of some secondary antibodies (Secondary Immunoreagents - Section 7.2, Summary of our LysoTracker and LysoSensor probes - Table 12.7) and anti-dye antibodies (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Anti-fluorophore antibodies and their conjugates - Table 7.19), as well as biotinylated enzymes and enzyme conjugates of NeutrAvidin biotin-binding protein 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), for use in diverse detection schemes.

Figure 7.36 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.

Figure 7.37 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 7.38 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 4.1 Comparison of the structures of D-biotin (top) and D-desthiobiotin (bottom).

Figure 7.100 Diagram illustrating the use of streptavidin agarose and a DSB-X biotin bioconjugate in affinity chromatography. A DSB-X biotin–labeled IgG antibody and its target antigen are used as an example.

Zenon Antibody Labeling Kits and Other Protein Labeling Kits

In addition to our extensive assortment of dye- and enzyme-conjugated secondary antibodies (Secondary Immunoreagents - Section 7.2), Molecular Probes has developed the important Zenon antibody labeling technology (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3), which utilizes a dye- or enzyme-labeled Fab fragment of an Fc-specific anti-IgG antibody to form stable complexes with the Fc portion of the corresponding mouse, rabbit, goat or human IgG antibody. Zenon labeling methods are rapid and quantitative and can permit use of multiple antibodies derived from the same species in a single multicolor experiment (Figure 7.56, , ).

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.

Kits for Labeling Proteins and Nucleic Acids - Section 1.2 describes several protein labeling kits that can be used to directly conjugate most of our proprietary dyes to antibodies and other proteins (Active esters and kits for labeling proteins and nucleic acids - Table 1.2, Molecular Probes' kits for protein and nucleic acid labeling - Table 1.3). Amine-reactive versions of all of the low molecular weight fluorophores that we use to prepare our conjugates, and their spectral properties and other characteristics, are extensively described Fluorophores and Their Amine-Reactive Derivatives - Chapter 1.

Gold Clusters and Magnetic Separation Media

In cooperation with Nanoprobes, Inc. (http://www.nanoprobes.com/), Molecular Probes offers NANOGOLD and Alexa Fluor FluoroNanogold 1.4 nm gold clusters covalently coupled to Fab' fragments of secondary antibodies (Secondary Immunoreagents - Section 7.2) or streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) for light and electron microscopy studies. The Captivate ferrofluid antibody and streptavidin conjugates (Secondary Immunoreagents - Section 7.2, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) and associated technology permit the magnetic separation of cells and cell components, and their visualization using a unique magnetic yoke and particle separation chamber (Figure 7.55).

Figure 7.55 Flow chart for the magnetic separation and analysis of a cell suspension. Cells are treated with an antibody or a biotinylated or DSB-X biotin–labeled probe that binds to cell-surface markers. The treated cells are incubated with the appropriate Captivate ferrofluid conjugates, which bind to target cells. The mixture is then transferred to a chamber that is inserted into a magnetic yoke. Under the influence of a strong magnetic field, the cells bound to Captivate ferrofluid conjugates are rapidly separated from the unbound cells. The separate cell populations can be analyzed by both fluorometry and fluorescence microscopy.

Molecular Probes prepares protein conjugates of a wide variety of fluorophores, most of which have been developed in our research laboratories, ranging from the blue-fluorescent Cascade Blue, Marina Blue and Alexa Fluor 350 dyes (Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores - Section 1.7) to the red-fluorescent Alexa Fluor 594, Texas Red and Texas Red-X dyes and the red- to infrared-fluorescent Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3). Zenon Antibody Labeling Kits are also available for antibody labeling with all of these dyes (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14). We also prepare antibody, streptavidin and NeutrAvidin biotin-binding protein conjugates of phycobiliproteins, as well as antibodies and streptavidin labeled with our tandem conjugates of the Alexa Fluor 610, Alexa Fluor 647 and Alexa Fluor 680 dyes with R-phycoerythrin (R-PE) (Phycobiliproteins - Section 6.4, Figure 6.34) and of allophycocyanin (APC) with the Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes (Phycobiliproteins - Section 6.4, Figure 6.37). These ternary conjugates of the phycobiliprotein, a low molecular weight fluorescence resonance energy transfer acceptor and a secondary detection reagent, are particularly useful for multicolor flow cytometry measurements using a single laser, such as the 488 nm spectral line of the argon-ion laser or the 633 nm spectral line of the He–Ne laser for excitation of R-PE tandem conjugates or APC tandem conjugates, respectively. The DyeMer dyes are bifluorophores that have intrinsically high Stokes shifts; they absorb maximally near 488 nm with fluorescence emissions at 611, 617 or 630 nm.

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.

Properties of the low molecular weight dyes that we use to prepare our conjugates are described in detail in Fluorophores and Their Amine-Reactive Derivatives - Chapter 1. In particular, we would like to highlight our:

• Alexa Fluor conjugates. Because of their superior brightness (Figure 7.1, Figure 7.36, Figure 7.37, Figure 7.38) and photostability (Figure 1.9, , , Figure 1.28, Figure 1.53), our Alexa Fluor conjugates are rapidly becoming the preferred reagents for all fluorescence-based immunoassays. Furthermore, with our simplified nucleic acid labeling technology (Labeling Oligonucleotides and Nucleic Acids - Section 8.2) and availability of new, longer-wavelength Alexa Fluor dyes, we anticipate significant use of the Alexa Fluor dyes for in situ hybridization applications in cells and on arrays (Detecting Nucleic Acid Hybridization - Section 8.5). We prepare a vast number of different conjugates from our spectrally distinct Alexa Fluor dyes: Alexa Fluor 350 (), Alexa Fluor 405 (), Alexa Fluor 430 (), Alexa Fluor 488 (), Alexa Fluor 500 (), 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 () and Alexa Fluor 750 () dyes, where the number refers to the near-optimal excitation wavelength for each dye. Our Alexa Fluor 488, Alexa Fluor 555 and Alexa Fluor 647 goat anti–mouse IgG antibody conjugates have significantly higher total fluorescence than do all the commercially available conjugates of the spectrally similar Cy2, Cy3 and Cy5 dyes that we have tested (Figure 7.1, Figure 7.36, Figure 7.37, Figure 7.38). These dyes and their properties are described in detail in Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3.
• Oregon Green conjugates. The Oregon Green 488 dye has excitation and emission spectra () that are virtually identical to those of fluorescein, yet offers greater photostability and a fluorescence signal that is essentially independent of pH above pH 6 (Figure 1.12). The Oregon Green 514 dye () is even more photostable than the Oregon Green 488 dye (Figure 1.46, Figure 7.23). The decreased tendency of the Oregon Green dyes to quench their fluorescence upon protein conjugation allows us to prepare conjugates that are more fluorescent than fluorescein conjugates (Figure 1.54). The available Oregon Green dyes are listed in Amine-reactive BODIPY dyes - Table 1.7 and discussed in Fluorescein, Oregon Green and Rhodamine Green Dyes - Section 1.5.
• BODIPY conjugates. We prepare a large number of reactive BODIPY dyes (BODIPY Dye Series - Section 1.4, Amine-reactive BODIPY dyes - Table 1.7), and one of these — the BODIPY FL dye — is an excellent substitute for fluorescein in some applications, although we generally recommend the Alexa Fluor 488 and Oregon Green 488 dyes for preparation of protein conjugates. Unlike fluorescein's fluorescence, the green fluorescence of BODIPY FL conjugates is pH independent. In addition, the BODIPY FL dye has an exceptionally narrow emission spectrum (), making it particularly useful for multicolor applications (Figure 1.43). Because the BODIPY FL dye is electrically neutral, BODIPY FL conjugates have proven useful for immunofluorescence studies in eosinophils, which contain positively charged eosinophil granule proteins that cause nonspecific binding of FITC-conjugated antibodies.
• Fluorescein conjugates. Although we feel that our Alexa Fluor 488, Oregon Green and BODIPY FL dyes will provide superior performance in most applications, we continue to provide high-quality fluorescein conjugates for researchers who prefer to use fluorescein in their applications. Molecular Probes has developed a reactive fluorescein derivative that typically yields conjugates with significantly greater fluorescence than other commercially available fluorescein-labeled proteins. Figure 1.54 shows the fluorescence intensity of IgG labeled in the traditional manner using FITC, compared with that of an IgG labeled using Molecular Probes' unique fluorescein-5-EX succinimidyl ester (F6130, BODIPY Dye Series - Section 1.4, ). Labeling with the fluorescein-5-EX reagent ensures that a greater signal is obtained for each IgG-bound fluorescein. Protein conjugates prepared from succinimidyl esters of fluorescein also have higher chemical stability than those prepared from fluorescein isothiocyanate (FITC).
• Rhodamine Red-X and Texas Red-X conjugates. Molecular Probes uses the succinimidyl esters of our Patented Rhodamine Red-X (R6160, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores - Section 1.7; , ) and Texas Red-X (T6134, T20175; Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores - Section 1.7; , ) fluorophores to prepare several detection reagents. The aminohexanoyl spacer ("X") apparently lessens the quenching that sometimes occurs when fluorescent dyes are conjugated to proteins. We have found that some of our Rhodamine Red-X and Texas Red-X protein conjugates are about twice as fluorescent as the corresponding conjugates prepared from Lissamine rhodamine B sulfonyl chloride and Texas Red sulfonyl chloride (Figure 1.76, Figure 1.83), thus providing a better signal-to-noise ratio. We continue to supply most of our original Texas Red conjugates for those customers who have developed protocols using these products. Conjugates of the Texas Red and Texas Red-X dyes () emit at wavelengths that have little overlap with the fluorescence of fluorescein () or R-phycoerythrin () and are particularly useful for multicolor applications. Alexa Fluor 568 () and Rhodamine Red-X () conjugates have maximal absorption at ~570 nm, making them the preferred probes for excitation by the 568 nm spectral line of the Ar–Kr laser used in some confocal laser-scanning microscopes.
• Long-wavelength Alexa Fluor conjugates. Our Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dye conjugates fill a need for bright and relatively photostable conjugates that can be excited by inexpensive, long-wavelength excitation sources such as the red He–Ne laser (633 nm) and red laser diodes. Excitation and detection at long wavelengths usually results in superior rejection of sample autofluorescence. We find the Alexa Fluor 635 dye to be the best fluorescent dye available for excitation by the red He–Ne laser at 633 nm and the 635 nm laser diode. Conjugates of the Alexa Fluor 647 dye have fluorescence that is superior to that of the spectrally similar Cy5 dye (Figure 1.25) on both proteins (Figure 7.38) and nucleic acids (Figure 8.50). The Alexa Fluor 680 dye has spectra virtually identical to those of the Cy5.5 dye (Figure 7.29), but its conjugates tend to be more fluorescent than those of the Cy5.5 dye. Our longest-wavelength Alexa Fluor dye — the Alexa Fluor 750 dye — has spectra similar to those of the Cy7 dye () and fluorescence emission that is well beyond essentially all biological autofluorescence. It may be possible to observe fluorescence of Alexa Fluor 750 conjugates in vivo because biological tissues are relatively transparent to excitation light in the 700–800 nm spectral range. Using these dyes, we have prepared conjugates of a number of proteins, as well as numerous Alexa Fluor phalloidin conjugates for staining F-actin filaments (Probes for Actin - Section 11.1, Spectral characteristics of our F-actin-selective probes - Table 11.1), the ULYSIS and ARES 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, Spectral characteristics of the fluorescent dyes available in Molecular Probes' ARES DNA Labeling Kits - Table 8.9) and the Alexa Fluor Oligonucleotide Amine Labeling Kits (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Oligonucleotide Amine Labeling Kits - Table 8.10). Zenon Antibody Labeling Kits are also available for antibody labeling with most of these dyes (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14). Conjugates of the Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes emit beyond the spectral range to which the human eye is sensitive. However, a filter combination that has been reported to be suitable for visual observation of Cy5 fluorescence should also be suitable observing for Alexa Fluor 647 conjugates. Conjugates of the Alexa Fluor 700 and Alexa Fluor 750 dyes may be difficult to detect without using red-enhanced photomultipliers or other suitable detection systems.
• Phycobiliprotein conjugates. In addition to our selection of immunoreagents labeled with organic dyes, Molecular Probes prepares phycobiliprotein-labeled secondary reagents (Phycobiliproteins - Section 6.4). The fluorescence yield of the red-fluorescent B- and R-phycoerythrin conjugates is theoretically equivalent to at least 30 fluorescein or 100 rhodamine molecules at comparable wavelengths. Because of the exceptional fluorescence and uniformly strong absorption for the phycoerythrins between 480 nm and 580 nm and allophycocyanin (APC) near 633 nm (Figure 6.32), phycobiliprotein-labeled detection reagents have been used extensively in flow cytometry to detect cell-specific expression of surface antigens. Researchers used a phycobiliprotein-conjugated antibody to detect interleukin-4 in a microplate assay and found that it was the only tested fluorophore that produced an adequate signal. Our streptavidin conjugate of R-phycoerythrin (SAPE; S866, S21388) has been extensively used to detect biotinylated nucleic acid probes on arrays (Figure 6.42) and is an important reagent for "tetramer technology" (MHC Tetramer Technology - Note 7.3 ). We also offer detection reagents labeled with APC () — one of the few fluorescent dyes that can be excited by the 633 nm spectral line of the He–Ne laser. In imaging applications, APC is both brighter and more photostable than the spectrally similar Cy5 dye (Figure 6.31). Phycobiliproteins - Section 6.4 discusses the spectral properties of phycobiliproteins in more detail. Preparation of phycobiliprotein-labeled mouse, rabbit and human primary antibodies is greatly simplified by availability of Zenon Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14) containing phycobiliprotein-derived labeling reagents.
• Tandem conjugates of phycobiliproteins. We have conjugated R-phycoerythrin (R-PE) with either the Alexa Fluor 610, Alexa Fluor 647 or Alexa Fluor 680 dye, then coupled these tandem dye derivatives to antibodies or streptavidin to yield secondary detection reagents (Tandem conjugates of R-phycoerythrin (R-PE) - Table 6.3) that can be excited with the 488 nm spectral line of the argon-ion laser (Phycobiliproteins - Section 6.4, Figure 6.34). Emission from the Alexa Fluor 610 conjugates of R-PE is at ~630 nm, which is a slightly longer wavelength than the emission of Texas Red dye–based tandem conjugates of R-PE (Figure 6.39). This slightly longer-wavelength emission maximum significantly improves the resolution that can be obtained when using the Alexa Fluor 610–R-PE tandem conjugates in place of Texas Red–R-PE tandem conjugates for multicolor flow cytometry. The exceptionally long-wavelength emission maximum of the Alexa Fluor 647–R-PE conjugates is at 667 nm and the energy transfer efficiency is very high (typically >98%), which results in low compensation in the red-orange fluorescence (R-PE) channel when the conjugates are used in combination with R-PE conjugates in multicolor flow cytometry applications (Figure 6.38). The Alexa Fluor 647–R-PE and Alexa Fluor 680–R-PE tandem conjugates have long-wavelength emission spectra that are virtually identical to those of Cy5 and Cy5.5 conjugates of R-PE, respectively, but fluorescence of our Alexa Fluor 647–R-PE bioconjugates is substantially greater than that of commercially available Cy5–R-PE streptavidin conjugate (Figure 6.39, Figure 6.40). These tandem conjugates can be used for simultaneous three- or four-color labeling with a single excitation (Figure 6.34) and are also extremely useful for multicolor applications, including immunohistochemistry and hybridization assays, that use excitation by a longer-wavelength excitation source such as the Nd:YAG laser (at 532 nm), green He–Ne laser (at 543 nm) or krypton-ion laser (at 568 nm). In addition to the tandem conjugates of R-PE, we have prepared conjugates of either the Alexa Fluor 680, Alexa Fluor 700 or Alexa Fluor 750 dye with APC and then conjugated these tandem dye derivatives to antibodies and streptavidin (Tandem conjugates of allophycocyanin (APC) - Table 6.4). The Alexa Fluor 680–APC ternary conjugates can be excited at 633–650 nm and their fluorescence detected separately from that of APC conjugates (Figure 6.37). Zenon Mouse IgG₁ Labeling Kits containing Fab fragments labeled with the tandem phycobiliprotein conjugates are currently available (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14).
• DyeMer conjugates. 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.
• Cascade Blue and Alexa Fluor 350 conjugates. Although less frequently used because of their spectral overlap with sample autofluorescence and their generally lower fluorescence yields, blue-fluorescent fluorophores remain important for multicolor applications such as fluorescence in situ hybridization (FISH) and polychromatic flow cytometry. Among the brightest UV light–excitable dyes are Molecular Probes' Patented Cascade Blue dyes and the Alexa Fluor 350 dye — a sulfonated derivative of 7-amino-4-methylcoumarin-3-acetic acid (AMCA). We have found that protein conjugates of the Alexa Fluor 350 dye are typically twice as fluorescent as AMCA conjugates (Figure 7.31). Furthermore, Alexa Fluor 350 conjugates have slightly shorter-wavelength emission () than AMCA conjugates (~442 nm versus ~448 nm), thus yielding better separation of their emission from that of fluorescein or the Alexa Fluor 488 dye. Conjugates of the Cascade Blue dye are intrinsically brighter than AMCA or Alexa Fluor 350 conjugates and have improved spectral resolution from the emission of fluorescein (Figure 1.100), an important advantage for multicolor applications. However, the shorter emission wavelength of the Cascade Blue conjugates () makes them appear less bright than Alexa Fluor 350 conjugates because of the limited spectral sensitivity of the human eye to the shorter-wavelength fluorescence of the Cascade Blue dye. Unlike many other fluorophores, such as fluorescein, the Cascade Blue dye resists quenching upon protein conjugation (Figure 7.32). Conjugates of the Cascade Blue dye (and the structurally similar Alexa Fluor 405 dye) are optimally excited by the 405 nm spectral line of the blue diode laser recently developed for fluorescence microscopy and flow cytometry.
• Marina Blue and Pacific Blue. Our Patented Marina Blue () and Pacific Blue () dyes, both of which are based on the 6,8-difluoro-7-hydroxycoumarin fluorophore (Figure 1.95, ), exhibit bright blue-fluorescent emission near 460 nm. The Marina Blue dyes are optimally excited by the intense 365 nm spectral line of the mercury-arc lamp, whereas the Pacific Blue dyes maximally absorb at ~415 nm. Conjugates of the Pacific Blue dye are optimally excited by the 405 nm spectral line of the blue diode laser recently developed for fluorescence microscopy and flow cytometry.
• Pacific Orange, Pacific Blue and Cascade Yellow conjugatesCascade Yellow conjugates. Our blue-fluorescent Alexa Fluor 405 () and Pacific Blue () dyes, yellow-fluorescent Cascade Yellow dye () and orange-fluorescent Pacific Orange dye () absorb maximally between 400 and 410 nm, making them near-perfect matches to the 405 nm spectral line of the violet laser recently developed for fluorescence microscopy and flow cytometry.
• CMNB-caged fluorescein conjugates. Our unique CMNB-caged fluorescein conjugates of the goat anti–mouse IgG and goat anti–rabbit IgG antibodies (G21061, G21080; Secondary Immunoreagents - Section 7.2) permit the fluorescent signal to be discriminated from background by photoactivation with ultraviolet light. At the same time, photoactivation creates a hapten for anti-fluorescein/Oregon Green antibody (Anti-Dye and Anti-Hapten Antibodies - Section 7.4) only at sites that are illuminated (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Figure 7.71), a process similar to photolithography.
• Fluorescent microspheres. Where they can be used, conjugates of our FluoSpheres and TransFluoSpheres polystyrene microspheres provide the greatest versatility in selection of wavelengths and other properties (Microspheres - Section 6.5, Figure 6.45). Not only are they intensely fluorescent, but TransFluoSpheres beads also have extremely large Stokes shifts (Figure 6.49). Fluorescent microspheres labeled with biotin, streptavidin, NeutrAvidin biotin-binding protein and protein A are described in Microspheres - Section 6.5 and Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6.
• Tyramide signal amplification (TSA) technology. TSA, described in Tyramide Signal Amplification (TSA) Technology - Section 6.2, yields extremely high signals at the binding site of horseradish peroxidase (HRP)–conjugated probes. The method (Figure 6.5) results in catalyzed deposition of one of our Alexa Fluor tyramide, Oregon Green 488 tyramide or Pacific Blue tyramide conjugates or of biotin-XX tyramide, DSB-X biotin tyramide or DNP-X tyramide. The biotin-XX tyramide or DSB-X tyramide that is deposited on the target can be detected with a fluorescent conjugate of avidin or 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) or can be further amplified with our ELF technology or a second round of TSA (Figure 6.5). Double amplification using TSA technology in combination with ELF technology permits ultrasensitive detection of low-abundance targets with high spatial resolution. HRP-conjugated antibodies that are useful for TSA can be rapidly and quantitatively prepared using our Zenon Horseradish Peroxidase Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14).
• Enzyme-Labeled Fluorescence (ELF) technology. Our ELF detection reagents and kits, which are described in detail in Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3, can be used to enhance the detection of biotinylated antibodies (Figure 6.21), biotinylated mRNA probes (Figure 8.93) and other haptenylated probes. When used in combination with alkaline phosphatase–streptavidin conjugates or alkaline phosphatase–labeled primary detection reagents, the ELF 97 phosphatase substrate yields a yellow-green–fluorescent precipitate at the site of enzymatic activity that is much more photostable than any simple dye-labeled antibody conjugates (Figure 6.16). The Zenon Alkaline Phosphatase Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14) facilitate the preparation of alkaline phosphatase–conjugated antibodies for use in combination with our ELF technology.
• Enzyme conjugates. Molecular Probes offers various secondary antibody, streptavidin and NeutrAvidin conjugates of alkaline phosphatase, horseradish peroxidase and β-galactosidase, which are all prepared by methods that result in an approximate 1:1 ratio of enzyme to carrier protein, thereby ensuring the retention of both carrier-protein binding and enzymatic activity (Alkaline phosphatase and horseradish peroxidase enzyme conjugates and Zenon Labeling Kits - Table 7.4). These conjugates are described in Secondary Immunoreagents - Section 7.2 and Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6. Our Zenon Alkaline Phosphatase and Horseradish Peroxidase Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14) permit the formation of enzyme-labeled mouse monoclonal antibodies using as little as submicrogram quantities of the primary antibody.
• Biotin and DSB-X biotin conjugates. Biotin and DSB-X biotin conjugates of secondary antibodies (Molecular Probes' biotinylated and desthiobiotinylated secondary antibodies - Table 7.11) permit the use of our fluorophore- and enzyme-labeled avidin, streptavidin and NeutrAvidin biotin-binding protein conjugates, our NANOGOLD and Alexa Fluor FluoroNanogold streptavidin and our Captivate ferrofluid avidin and streptavidin products (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6), as well as the TSA and ELF amplification technologies (Tyramide Signal Amplification (TSA) Technology - Section 6.2, Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3) in combination with immunolabeling methods. Binding of the DSB-X biotin derivatives to avidin- and streptavidin-labeled targets is fully reversible under very mild conditions (Figure 7.100, Figure 7.104). See Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6 for a description of our unique DSB-X biotin technology.
• Signal amplification kits. Using antibody conjugates of our intensely fluorescent Alexa Fluor 488 dye, Molecular Probes has developed the Alexa Fluor 488 Signal Amplification Kit (A11053, Secondary Immunoreagents - Section 7.2), which gives a greater than 10-fold amplification of the green fluorescence of fluorescein-labeled antibodies (Figure 7.49) while at the same time considerably increasing the photostability of the stained sample. Similar kits have been developed that yield exceptionally intense green, red-orange or red fluorescence from any mouse antibody. These kits are described in Secondary Immunoreagents - Section 7.2.

Figure 7.1 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 1.28 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 1.53 Photobleaching profiles of cells stained with Alexa Fluor 488 or fluorescein conjugates of goat anti–mouse IgG antibody F(ab')₂ fragment (A11017, F11021) were used to detect HEp-2 cells probed with human anti-nuclear antibodies. Samples were continuously illuminated and images were collected every 5 seconds with a cooled CCD camera. Normalized intensity data demonstrate the difference in photobleaching rates.




Figure 1.43 Normalized fluorescence emission spectra of goat anti–mouse IgG antibody conjugates of fluorescein (FL), tetramethylrhodamine (TMR) and the Texas Red (TR) dyes, shown by dashed lines (---), as compared with goat anti–mouse IgG antibody conjugates of BODIPY FL, BODIPY TMR and BODIPY TR dyes, respectively, shown by solid lines (—).




Figure 1.46 Comparison of photostability of green-fluorescent antibody conjugates. The following fluorescent goat anti–mouse IgG antibody conjugates were used to detect mouse anti–human IgG antibody labeling of human anti-nuclear antibodies in HEp-2 cells on prefixed test slides (INOVA Diagnostics Corp.): Oregon Green 514 (O6383, ), Alexa Fluor 488 (A11001, ), BODIPY FL (B2752, ), Oregon Green 488 (O6380, ) or fluorescein (F2761, ). Samples were continuously illuminated and viewed on a fluorescence microscope using a fluorescein longpass filter set. Images were acquired every 5 seconds. For each conjugate, three data sets, representing different fields of view, were averaged and then normalized to the same initial fluorescence intensity value to facilitate comparison.





Figure 7.23 Comparison of the photostability of immunofluorescent staining by Oregon Green 514 goat anti–mouse IgG antibody (O6383, upper series) and by fluorescein goat anti–mouse IgG antibody (F2761, lower series). Bovine pulmonary arterial endothelial cells were fixed with formaldehyde and permeabilized in cold acetone. Following blocking in 1% BSA, 1% normal goat serum, 0.1% Tween 20 in PBS, samples were incubated for one hour with 60 µg/mL mouse monoclonal anti–human cytochrome oxidase subunit I antibody (A6403, Primary Antibodies for Diverse Applications - Section 7.5), after which they were rinsed and incubated with fluorescent anti–mouse IgG secondary antibodies at 10 µg/mL for 30 minutes. Samples were continuously illuminated and viewed on a fluorescence microscope using an Omega Optical fluorescein longpass filter set, a Star 1 CCD camera (Photometrics) and Image-1 software (Universal Imaging Corp.). Images acquired 0, 20, 40 and 90 seconds after the start of illumination (as indicated in the top left-hand corner of each panel) clearly demonstrate the superior photostability of the Oregon Green 514 conjugate.





Figure 1.54 Comparison of relative fluorescence as a function of the number of fluorophores attached per protein for goat anti–mouse IgG antibody conjugates prepared using Oregon Green 514 carboxylic acid succinimidyl ester (O6139, ), Oregon Green 488 carboxylic acid succinimidyl ester (O6147, ), fluorescein-5-EX succinimidyl ester (F6130, ) and fluorescein isothiocyanate (FITC, F143, F1906, F1907, ). 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.



Figure 1.83 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates of Texas Red-X succinimidyl ester (T6134, ) and Texas Red sulfonyl chloride (T353, ). Conjugate fluorescence was 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. Higher numbers of fluorophores attached per protein are attainable with the Texas Red-X dye due to the lesser tendency of this dye to induce protein precipitation.





Figure 1.76 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates of Rhodamine Red-X succinimidyl ester (R6160, ) and Lissamine rhodamine B sulfonyl chloride (L20, L1908, ). 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. Higher numbers of fluorophores attached per protein are attainable with Rhodamine Red-X dye due to the lesser tendency of this dye to induce protein precipitation.




Figure 6.16 Photostability comparison for ELF 97 alcohol– and fluorescein-labeled tubulin preparations. Tubulin in acetone-fixed CRE BAG 2 mouse fibroblasts was labeled with an anti–β-tubulin monoclonal antibody and then detected using biotin-XX goat anti–mouse IgG antibody (B2763) in conjunction with either our ELF 97 Cytological Labeling Kit (E6603, ) or fluorescein streptavidin (S869, ). Alternatively, anti-tubulin labeling was detected directly using fluorescein goat anti–mouse IgG antibody (F2761, ). The photostability of labeling produced by the three methods was compared by continuously illuminating stained samples on a fluorescence microscope using Omega Optical longpass optical filter sets. Images were acquired every 5 seconds using a Star 1 CCD camera (Photometrics); the average fluorescence intensity in the field of view was calculated with Image-1 software (Universal Imaging Corp.) and expressed as a fraction of the initial intensity. Three data sets, representing different fields of view, were averaged for each conjugate to obtain the plotted time courses.




Figure 6.21 Schematic diagram of the method employed in our ELF 97 mRNA In Situ Hybridization (E6604, E6605), Cytological Labeling (E6603) and Immunohistochemistry (E6600) Kits. Samples are probed with haptenylated or biotinylated target-specific probes such as antibodies or hybridization probes. Next, alkaline phosphatase conjugates of streptavidin or the hapten-specific probe are applied. Alternatively, a biotinylated antibody and biotinylated alkaline phosphatase can be used with standard bridging methods to increase the penetration in tissue, a method that is employed in our ELF 97 Immunohistochemistry Kit. The sample is then incubated with the ELF 97 phosphatase substrate, which forms an intense yellow-green–fluorescent ELF 97 alcohol precipitate at the site of alkaline phosphatase activity.




Figure 8.93 Schematic representation of mRNA in situ hybridization detection using the Enzyme-Labeled Fluorescence (ELF) technology (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3). Alkaline phosphatase converts ELF 97 phosphate (black triangles) to a brilliant green-fluorescent precipitate (green squares).





Figure 6.31 A comparison of the photobleaching rates of APC and Cy5 conjugates. The microtubules of bovine pulmonary artery endothelial cells were stained with mouse anti–α-tubulin antibody (A11126) in combination with goat anti–mouse IgG labeled antibody with either crosslinked APC (A865, top series) or the Cy5 dye (bottom series). The samples were exposed to continuous illumination, and the images were acquired at 30-second intervals with a Quantex cooled CCD camera (Photometrics) using filter sets appropriate for both APC and Cy5 dye.




Figure 6.32 Normalized absorption spectra for B-PE, R-PE and APC.




Figure 6.38 Fluorescence emission spectra of Alexa Fluor 647–R-phycoerythrin streptavidin (S20992; red) and Cy5–R-phycoerythrin streptavidin (Caltag Laboratories; blue) tandem conjugates. Panel A shows a comparison of the spectra on a relative fluorescence intensity scale for samples prepared with equal absorbance at the excitation wavelength (488 nm). Panel B shows the same data normalized to the same peak intensity value to facilitate comparison of the spectral profiles.




Figure 6.39 Fluorescence emission spectra of Alexa Fluor 610–R-phycoerythrin streptavidin (S20982; red) and Texas Red–R-phycoerythrin streptavidin (Caltag Laboratories; blue) tandem conjugates. Panel A shows a comparison of the spectra on a relative fluorescence intensity scale for samples prepared with equal absorbance at the excitation wavelength (488 nm). Panel B shows the same data normalized to the same peak intensity value to facilitate comparison of the spectral profiles.




Figure 6.40 Comparison of immunofluorescent staining by R-phycoerythrin–dye tandem conjugates. EL4 cells labeled with a biotinylated anti-CD44 monoclonal antibody (Caltag Laboratories) were detected with streptavidin conjugates of Alexa Fluor 647–R-PE (S20992) or Cy5–R-PE (Serotec). The cells were analyzed by flow cytometry on a Coulter XL cytometer using excitation at 488 nm. Data were obtained using an bandpass emission filter (675 ± 20 nm; upper panels) or a longpass emission filter (>650 nm; lower panels). In each histogram, unstained and stained cells are represented by the blue and red lines, respectively. The numbers above each peak represent mean channel fluorescence intensities. Data provided by William Telford, NCI-NIH, Bethesda, MD.





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.





Figure 6.42 R-phycoerythrin used to detect DNA on a microarray. A DNA microarray containing a decreasing dilution of calf thymus DNA was hybridized with a biotinylated DNA probe and then incubated with R-phycoerythrin–streptavidin (SAPE; S866, S21388). After washing, the fluorescence signal was detected on a Packard ScanArray 5000 using three different detection configurations: 488 nm excitation (argon-ion laser)/570 nm emission filter (left); 543.5 nm excitation (He–Ne laser)/570 nm emission filter (middle); 543.5 nm excitation (He–Ne laser)/592 nm emission filter (right).




Figure 6.45 Normalized fluorescence emission spectra of our FluoSpheres beads, named according to their excitation/emission maxima (nm): 1) blue (365/415), 2) blue (350/440), 3) yellow-green (505/515), 4) orange (540/560), 5) red-orange (565/580), 6) red (580/605), 7) crimson (625/645), 8) dark red (660/680), 9) far-red (690/720) and 10) infrared (715/755) FluoSpheres beads.





Figure 6.49 Schematic diagram of the advantages of the large Stokes shift exhibited by our TransFluoSpheres beads. A1 and E1 represent the absorption and emission bands of a typical TransFluoSpheres bead. The large separation of the absorption and emission maxima (Stokes shift) is characteristic of our TransFluoSpheres beads. Unlike most low molecular weight fluorescent dyes, which show considerable overlap of their absorption and emission spectra, the TransFluoSpheres beads can be excited (EX) across the entire absorption band A1 and the resulting fluorescence can be detected across the full emission band E1, thereby allowing the researcher to maximize the signal (S1). Moreover, because of the large Stokes shifts of the TransFluoSpheres beads, researchers can often avoid problems associated with autofluorescence. The absorption and emission bands of a typical autofluorescent component are represented in this Figure .y A2 and E2. Although the endogenous fluorescent species will be excited simultaneously with the TransFluoSpheres beads, the resulting emission (E2) does not coincide with E1 and is therefore readily rejected by suitably chosen optical filters.

Figure 7.104 Cell separation using Captivate ferrofluid streptavidin and DSB-X biotin conjugates. A mixed population of cells is first mixed with a DSB-X biotin–labeled antibody against an appropriate surface antigen (panel A); subsequent incubation results in the labeling of a specific subpopulation (panel B). The sample is then incubated with Captivate ferrofluid streptavidin (C21476), which binds to the DSB-X biotin hapten, allowing the labeled cells to be isolated via a magnetic field (panel C). After the unlabeled cells have been washed away, the captured cells can be released by reversing the streptavidin linkage to DSB-X biotin with unlabeled biotin (panel D).

Figure 7.49 Antibody amplification scheme using our superior Alexa Fluor conjugates, permitting enhanced detection of mouse primary antibodies. Molecular Probes offers three Alexa Fluor Signal Amplification Kits for Mouse Antibodies containing antibody conjugates of the Alexa Fluor 488 (A11054), Alexa Fluor 568 (A11066) and Alexa Fluor 594 (A11067) dyes, which yield green, red-orange and red fluorescence, respectively. These kits each use two Alexa Fluor conjugates to detect antibodies derived from mouse. An Alexa Fluor rabbit anti–mouse IgG antibody conjugate is first used to bind to the mouse primary antibody. The fluorescence signal is then dramatically enhanced by the addition of an Alexa Fluor conjugate of goat anti–rabbit IgG antibody.

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 7.29 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 8.50 Fluorescence emission spectra of single-stranded DNA labeled with equivalent levels of the Alexa Fluor 647 dye (blue curve) or Cy5 dye (red curve) using aminoallyl dUTP incorporation followed by incubation with a reactive dye. While the absorbance for the two samples is similar, the fluorescence emission from the Alexa Fluor 647 dye–labeled DNA is several times more intense than that of the Cy5 dye–labeled DNA.

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 1.100 Normalized fluorescence emission spectra of Cascade Blue (CB), 7-amino-4-methylcoumarin (AMC) and fluorescein in aqueous solutions.

Figure 7.32 Histograms showing the fluorescence per fluorophore for A) fluorescein and B) Cascade Blue conjugated to various proteins, relative to the fluorescence of the free dye in aqueous solution, represented by 100 on the y-axis. The proteins represented are: 1) avidin, 2) bovine serum albumin, 3) concanavalin A, 4) goat IgG, 5) ovalbumin, 6) protein A, 7) streptavidin and 8) wheat germ agglutinin.

Figure 1.95 Comparison of the pH-dependent fluorescence changes produced by attachment of electron-withdrawing fluorine atoms to a hydroxycoumarin. 7-Hydroxy-4-methylcoumarin-3-acetic acid (, H1428) and 6,8-difluoro-7-hydroxy-4-methylcoumarin (, D6566) demonstrate the decrease of the pK_a from ~7.4 to ~6.2. Fluorescence intensities were measured for equal concentrations of the two dyes using excitation/emission at 360/450 nm.

Figure 7.71 Schematic representation of photoactivated fluorescence combined with sample masking. Initially, no fluorescence is observed from samples stained with a CMNB-caged fluorescein-labeled secondary detection reagent (panel A). The desired mask is then placed over the sample (panel B), after which the sample is exposed to UV light. The mask is then removed; fluorescein molecules present in the unmasked portion of the sample are uncaged by the UV light and fluoresce brightly when viewed with the appropriate filters (panel C). Uncaged fluorescein may now also serve as a hapten for further signal amplification using our anti-fluorescein/Oregon Green antibody (A889). For example, probing with the anti-fluorescein/Oregon Green antibody followed by staining with the Alexa Fluor 594 goat anti–mouse IgG antibody (A11005) can be used to change the color of the uncaged probe to red fluorescent (panel D).