7.1 A Wide Variety of Protein Conjugates

7.1 A Wide Variety of Protein Conjugates Overview of Molecular Probes Protein Conjugates 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 and the dye-to-protein ratio (DOS). 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 7.44, Figure 7.45, Figure 7.46), yet have low background and often have better spectral resolution. Moreover, all of our primary and secondary antibody conjugates and our avidin, streptavidin, NeutrAvidin and CaptAvidin biotinbinding protein conjugates are tested on cell samples to ensure low nonspecific binding and high specific staining. Table 7.3, Table 7.7 and Table 7.17 list our current offerings of fluorescent secondary immunoreagents and avidins. We also offer biotin, DSB-X biotin (a readily reversible version of biotin; Figure 7.89, Figure 7.90) and enzyme conjugates of some secondary antibodies (Section 7.3, Table 7.6) and anti-dye antibodies (Section 7.4, Table 7.13), as well as biotinylated enzymes and enzyme conjugates of NeutrAvidin biotin-binding protein and streptavidin (Section 7.6, Table 7.17), for use in diverse detection schemes. Zenon One Technology and Protein Labeling Kits In addition to our extensive assortment of dye- and enzymeconjugated secondary antibodies (Section 7.3), Molecular Probes has developed the important Zenon One technology (Section 7.2), which utilizes a dye- or enzyme-labeled Fab fragment of goat anti mouse IgG 1 Fc antibody to form stable complexes of the Fc portion of any mouse IgG 1 antibody. Zenon One labeling methods are rapid and quantitative and can permit use of more than one mouse monoclonal antibody in a single multicolor experiment (Figure 7.32, Figure 7.37, Figure 7.39). 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 (Table 1.1, Table 1.2). 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 Chapter 1. Gold Clusters and Magnetic Separation Media In cooperation with Nanoprobes, Inc., Molecular Probes offers NANOGOLD and Alexa Fluor FluoroNanogold 1.4 nm gold clusters covalently coupled to Fab fragments of secondary antibodies (Section 7.3) or streptavidin (Section 7.6) for light and electron microscopy studies. The Captivate ferrofluid antibody and streptavidin conjugates (Section 7.3, Section 7.6) and associated technology permit the magnetic separation of cells and cell components, and their visualization using a unique yoke and particle separation chamber (Figure 7.62). Our Exceptional Fluorophores 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 (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 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes (Section 1.3). Zenon One Mouse IgG 1 Labeling Kits are also available for antibody labeling with all of these dyes (Section 7.2, Table 7.1). We also prepare antibody, (strept)avidin 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) (Section 6.4, Figure 6.31) and of allophycocyanin (APC) with the Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes (Section 6.4, Figure 6.34). 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 cytometric 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. Properties of the low molecular weight dyes that we use to prepare our conjugates are described in detail in Chapter 1. In particular, we would like to highlight our: Alexa Fluor conjugates. Because of their superior brightness (Figure 7.1, Figure 7.44, Figure 7.45, Figure 7.46) and photostability (Figure 1.48, Figure 7.2), our Alexa Fluor conjugates Figure 7.1 Flow cytometry was used to compare the brightness of Molecular Probes Alexa Fluor 647 goat anti mouse IgG antibody (red, A-21235) 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 long-pass emission filter (>650 nm). Section 7.1 189

are rapidly becoming the preferred reagents for all fluorescence-based immunoassays. Furthermore, with our simplified nucleic acid labeling technology (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 1 (Section 8.5). We prepare a vast number of different conjugates from 14 spectrally distinct Alexa Fluor dyes: Alexa Fluor 350 (Figure 7.3), Alexa Fluor 430 (Figure 7.4), Alexa Fluor 488 (Figure 7.5), Alexa Fluor 532 (Figure 7.6), Alexa Fluor 546 (Figure 7.7), Alexa Fluor 555 (Figure 7.8), Alexa Fluor 568 (Figure 7.9), Alexa Fluor 594 (Figure 7.10), Alexa Fluor 633 (Figure 7.11), Alexa Fluor 647 (Figure 7.12), Alexa Fluor 660 (Figure 7.13), Alexa Fluor 680 (Figure 7.14), Alexa Fluor 700 (Figure 7.15) and Alexa Fluor 750 (Figure 7.16) dyes, where the number refers to the near-optimal excitation wavelength for each of the dyes. 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 7.44, Figure 7.45, Figure 7.46). At this time we only offer the Alexa Fluor 610 dye in the form of tandem conjugates of R-PE (Section 6.4, Table 6.3). These dyes and their properties are described in detail in Section 1.3. Oregon Green conjugates. The Oregon Green 488 dye has excitation and emission spectra (Figure 7.17) 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 1.11). The Oregon Green 514 dye (Figure 7.18) is even more Figure 7.2 A comparison of the photobleaching rates of two Alexa Fluor dyes and the well-known fluorescein and Cy3 fluorophores. The cytoskeleton of bovine pulmonary artery endothelial cells (BPAEC) was labeled with (left series) Alexa Fluor 488 phalloidin (A-12379) and mouse monoclonal anti α-tubulin antibody (A-11126) in combination with Alexa Fluor 546 goat anti mouse IgG antibody (A-11003) or (right series) fluorescein phalloidin (F-432) and anti α-tubulin antibody in combination with a commercially available Cy3 goat anti mouse IgG antibody. The pseudocolored images are the first, second, fourth and eighth taken at 30 second intervals (0, 30, 90, and 210 seconds of exposure). The images were acquired sequentially with fluorescein and rhodamine bandpass filter sets. Figure 7.3 Fluorescence excitation and emission spectra of Alexa Fluor 350 goat anti mouse IgG antibody (A-11045) in ph 8.0 buffer. Figure 7.4 Absorption and fluorescence emission spectra of Alexa Fluor 430 goat anti mouse IgG antibody (A-11063) in ph 7.2 buffer. Custom conjugations of our Alexa Fluor dyes and many other dyes are available; contact custom@probes.com. Figure 7.5 Absorption and fluorescence emission spectra of Alexa Fluor 488 goat anti mouse IgG antibody (A-11001) in ph 8.0 buffer. Figure 7.6 Absorption and fluorescence emission spectra of Alexa Fluor 532 goat anti mouse IgG antibody (A-11002) in ph 7.2 buffer. 190 Chapter 7 Antibodies, Avidins, Lectins and Related Products www.probes.com

Figure 7.7 Absorption and fluorescence emission spectra of Alexa Fluor 546 goat anti mouse IgG antibody (A-11003) in ph 7.2 buffer. Figure 7.8 Absorption and fluorescence emission spectra of Alexa Fluor 555 goat anti mouse IgG antibody (A-21422) in ph 7.2 buffer. Figure 7.9 Absorption and fluorescence emission spectra of Alexa Fluor 568 goat anti mouse IgG antibody (A-11004) in ph 7.2 buffer. Figure 7.10 Absorption and fluorescence emission spectra of Alexa Fluor 594 goat anti mouse IgG antibody (A-11005) in ph 7.2 buffer. Figure 7.11 Fluorescence excitation and emission spectra of Alexa Fluor 633 goat anti mouse IgG antibody (A-21050) in ph 7.2 buffer. Figure 7.12 Absorption and fluorescence emission spectra of Alexa Fluor 647 goat anti mouse IgG antibody (A-21235) in ph 7.2 buffer. Figure 7.13 Absorption and fluorescence emission spectra of Alexa Fluor 660 goat anti mouse IgG antibody (A-21054) in ph 7.2 buffer. Figure 7.14 Absorption and fluorescence emission spectra of Alexa Fluor 680 goat anti mouse IgG antibody (A-21057) in ph 7.2 buffer. Figure 7.15 Absorption and fluorescence emission spectra of Alexa Fluor 700 goat anti mouse IgG antibody (A-21036) in ph 7.2 buffer. Figure 7.16 Absorption and fluorescence emission spectra of Alexa Fluor 750 goat anti mouse IgG antibody (A-21037) in ph 7.2 buffer. Figure 7.17 Absorption and fluorescence emission spectra of Oregon Green 488 goat anti mouse IgG antibody (O-6380) in ph 8.0 buffer. Figure 7.18 Absorption and fluorescence emission spectra of Oregon Green 514 goat anti mouse IgG antibody (O-6383) in ph 8.0 buffer. Section 7.1 191

Figure 7.20 Absorption and fluorescence emission spectra of BODIPY FL goat anti mouse IgG antibody (B-2752) in ph 7.2 buffer. Figure 7.21 Absorption and fluorescence emission spectra of Rhodamine Red-X goat anti mouse IgG antibody (R-6393) in ph 8.0 buffer. photostable than the Oregon Green 488 dye (Figure 1.42, Figure 1.57, Figure 7.19, Figure 11.8). 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 1.49). The Oregon Green dyes are listed in Table 1.4 and discussed in Section 1.5. BODIPY conjugates. We prepare a large number of reactive BODIPY dyes (Section 1.4, Table 1.4), 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 (Figure 7.20), making it particularly useful for multicolor applications (Figure 1.39). 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. 2 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 1.49 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 (F-6130, Section 1.4, Figure 1.54). 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). 3 Rhodamine Red-X and Texas Red-X conjugates. Molecular Probes uses the succinimidyl esters of our patented Rhodamine Red-X (R-6160, Section 1.7; Figure 1.70, Figure 7.21) and Texas Red-X (T-6134, T-20175; Section 1.7; Figure 1.77, Figure 7.22) 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 Figure 7.19 Photostability comparison of Oregon Green 514 phalloidin (O-7465, upper series) and fluorescein phalloidin (F-432, lower series). CRE BAG 2 fibroblasts were fixed with formaldehyde, then permeabilized with acetone and stained with the fluorescent phallotoxin. Samples were illumi- nated continuously and viewed on a fluorescence microscope equipped with a fluorescein longpass optical filter set. Images acquired at 1, 10, 20 and 30 seconds after the start of illumination (left to right) demonstrate the superior photostability of the Oregon Green 514 fluorophore. 192 Chapter 7 Antibodies, Avidins, Lectins and Related Products www.probes.com

conjugates prepared from Lissamine rhodamine B sulfonyl chloride and Texas Red sulfonyl chloride 4 (Figure 1.71, Figure 1.78), 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 (Figure 7.22) emit at wavelengths that have little overlap with the fluorescence of fluorescein (Figure 7.23) or R-phycoerythrin (Figure 7.24) and are particularly useful for multicolor applications. 5 7 Alexa Fluor 568 (Figure 7.9) and Rhodamine Red-X (Figure 7.21) 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 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. Conjugates of the Alexa Fluor 647 dye have fluorescence that is superior to that of the spectrally similar Cy5 dye (Figure 1.22) both on proteins (Figure 7.46) and nucleic acids (Figure 7.25). The Alexa Fluor 680 dye has spectra virtually identical to those of the Cy5.5 dye (Figure 7.26), 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 (Figure 7.16) 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 (Section 11.1, Table 11.1), the ULYSIS and ARES Nucleic Acid Labeling Kits (Section 8.2; Table 8.7, Table 8.8) and the Alexa Fluor Oligonucleotide Amine Labeling Kits (Section 8.2, Table 8.9). Zenon One Mouse IgG 1 Labeling Kits are available for all of these dyes (Section 7.2, Table 7.1). 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 8 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. Figure 7.22 Fluorescence excitation and emission spectra of Texas Red-X goat anti mouse IgG antibody (T-6390) in ph 7.2 buffer. Figure 7.23 Absorption and fluorescence emission spectra of fluorescein goat anti mouse IgG antibody (F-2761) in ph 8.0 buffer. Figure 7.24 Absorption and fluorescence emission spectra of R-phycoerythrin (P-801) in ph 7.5 buffer. Figure 7.25 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 7.26 Comparison of the fluorescence spectra of the Alexa Fluor 680 and Cy5.5 dyes. Spectra have been normalized to the same intensity for comparison purposes. The Texas Red dyes originated while Molecular Probes was located in Plano, Texas, from 1978 1982. Section 7.1 193

Figure 7.27 Absorption and fluorescence emission spectra of allophycocyanin (A-803) in ph 7.5 buffer. Figure 7.28 Comparison of the relative fluorescence of 7-amino-4-methylcoumarin-3-acetic acid (AMCA) streptavidin ( ) and Alexa Fluor 350 streptavidin, a sulfonated AMCA derivative (S-11249, ). 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 7.30 Absorption and fluorescence emission spectra of Marina Blue goat anti mouse IgG antibody (M-10991) in ph 8.0 buffer. Phycobiliprotein conjugates. In addition to our selection of immunoreagents labeled with organic dyes, Molecular Probes prepares phycobiliprotein-labeled secondary reagents (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 their exceptional fluorescence and uniformly strong absorption for the phycoerythrins between 480 580 nm and allophycocyanin (APC) near 633 nm (Figure 6.29), phycobiliprotein-labeled detection reagents have been used extensively in flow cytometry to detect cell-specific expression of surface antigens. 9 11 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. 12 Our streptavidin conjugate of R-phycoerythrin (S-866, S-21388) has been extensively used to detect biotinylated nucleic acid probes on arrays 13 18 (Figure 6.39) and is an important reagent for tetramer technology (see MHC Tetramer Technology in Section 6.4). We also offer detection reagents labeled with APC (Figure 7.27) one of the few fluorescent dyes that can be excited by the 633 nm spectral line of the He Ne laser. 19 APC is both brighter and more photostable than the spectrally similar Cy5 dye (Figure 6.28). Section 6.4 discusses the spectral properties of phycobiliproteins in more detail. Preparation of phycobiliprotein-labeled monoclonal antibodies is greatly simplified by availability of Zenon One Mouse IgG 1 Labeling Kits (Section 7.2; Table 7.1) 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 (Table 6.3) that can be excited with the 488 nm spectral line of the argon-ion laser (Section 6.4). 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 20 23 (Figure 6.36). The exceptionally longwavelength 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 6.35). 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 conjugates (Figure 6.35, Figure 6.37). These tandem conjugates can be used for simultaneous three- or four-color labeling with a single excitation (Figure 6.31) and are also extremely useful for multicolor applications, including immunohistochemistry and hybridization-based assays, that use excitation by longerwavelength excitation sources 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 (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 6.34). Zenon One Mouse IgG 1 Labeling Kits for the ternary phycobiliprotein dye derivative conjugates with Alexa Fluor dyes and secondary detection reagents are currently under development at Molecular Probes. 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. Among the brightest UV light excitable dyes are Molecular Probes patented Cascade Blue dyes 24 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 7.28). Furthermore, Alexa Fluor 350 conjugates have slightly shorter-wavelength emission (Figure 194 Chapter 7 Antibodies, Avidins, Lectins and Related Products www.probes.com

7.3) 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 25 or Alexa Fluor 350 conjugates and have improved spectral resolution from the emission of fluorescein (Figure 1.86), an important advantage for multicolor applications. However, the shorter emission wavelength of the Cascade Blue conjugates (Figure 1.87) 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 7.29). Marina Blue and Pacific Blue conjugates. Our patented Marina Blue (Figure 7.30) and Pacific Blue (Figure 7.31) dyes, both of which are based on the 6,8-difluoro-7- hydroxycoumarin fluorophore 26 (Figure 1.91, Figure 1.92) 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. CMNB-caged fluorescein conjugates. Our unique CMNB-caged fluorescein conjugates of the goat anti mouse IgG and goat anti rabbit IgG antibodies (G-21061, G-21080; Section 7.3) and of streptavidin (S-21380, Section 7.6) 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 (Section 7.4) only at sites that are illuminated (Figure 7.93), 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 27,28 (Section 6.5, Figure 6.42). Not only are they intensely fluorescent, but TransFluoSpheres beads also have extremely large Stokes shifts (Figure 6.46). Fluorescent microspheres labeled with biotin, streptavidin, NeutrAvidin biotin-binding protein and protein A are described in Section 7.6 and Section 6.5. Tyramide signal amplification (TSA) technology. TSA is described in Section 6.2, yields extremely high signals at the site of binding of horseradish peroxidase conjugated probes. The method (Figure 6.6) 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 (Section 7.6, Table 7.17) or can be further amplified with our ELF technology 29 or a second round of TSA (Figure 6.6). Double amplification by TSA in combination with ELF permits ultrasensitive detection of low-abundance targets with high spatial resolution. 29 HRP conjugates that are useful for TSA can be rapidly and quantitative prepared from any mouse IgG 1 isotype antibody using our Zenon One Horseradish Peroxidase Mouse IgG 1 Labeling Kit (Z-25054, Section 7.2). Enzyme-Labeled Fluorescence (ELF) technology. Our ELF detection reagents and kits, which are described in detail in Section 6.3, can be used to enhance the detection of biotinylated antibodies (Figure 6.21), biotinylated mrna probes (Figure 8.84) 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 6.17). The Zenon One Alkaline Phosphatase Mouse IgG 1 Labeling Kit (Z-25050, Section 7.2) facilitates the preparation of alkaline phosphatase conjugates of any mouse IgG 1 antibody 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 carrierprotein binding and enzymatic activity (Table 7.6). These conjugates are described in Figure 7.29 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 7.31 Absorption and fluorescence emission spectra of Pacific Blue goat anti mouse IgG antibody (P-10993) in ph 8.0 buffer. Molecular Probes offers 36 TSA Kits with an extensive assortment of dyes and haptens see Section 6.2. Section 7.1 195

Our Zenon Labeling Kits with R-phycoerythrin (R-PE) and allophycocyanin (APC), as well as the tandem conjugates of R-PE and APC with the longer wavelength Alexa Fluor dyes, make it possible to quantitatively label mouse IgG 1 antibodies, to use multiple mouse antibodies in the same protocol and to label submicrograms of a primary antibody in the presence of other non-antibody proteins. See Section 7.2 for an extensive description of our Zenon labeling technology. Section 7.3 and Section 7.6. Our Zenon One Alkaline Phosphatase and Horseradish Peroxidase Mouse IgG 1 Labeling Kits (Z-25050, Z-25054; Section 7.2) permit the formation of enzyme-labeled mouse monoclonal antibodies using as little as submicrograms of the primary antibody. Biotin and DSB-X biotin conjugates. Biotin and DSB-X biotin conjugates of secondary antibodies (Table 7.10) permit the use of our fluorescent dye, enzyme, Alexa Fluor FluoroNanogold and NANOGOLD 1.4 nm gold cluster and Captivate ferrofluid avidin and streptavidin products (Section 7.6) and our TSA and ELF amplification technologies (Section 6.2, 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 7.89, Figure 7.94). See 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 (A-11053, Section 7.3) that gives a greater than tenfold amplification of the green fluorescence of fluorescein-labeled antibodies (Figure 7.55), 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 Section 7.3. References 1. J Histochem Cytochem 47, 1179 (1999); 2. J Immunol Methods 217, 113 (1998); 3. Bioconjug Chem 6, 447 (1995); 4. Bioconjug Chem 7, 482 (1996); 5. Acta Histochem 89, 85 (1990); 6. Proc Natl Acad Sci U S A 85, 3546 (1988); 7. Cell Biophys 7, 129 (1985); 8. J Histochem Cytochem 48, 437 (2000); 9. J Cell Biol 116, 1291 (1992); 10. J Immunol Methods 149, 159 (1992); 11. Proc Natl Acad Sci U S A 85, 4672 (1988); 12. J Immunol Methods 128, 109 (1990); 13. J Biol Chem 276, 27042 (2001); 14. Proc Natl Acad Sci U S A 98, 8862 (2001); 15. Proc Natl Acad Sci U S A 97, 3260 (2000); 16. Proc Natl Acad Sci U S A 97, 2680 (2000); 17. Proc Natl Acad Sci U S A 95, 3752 (1998); 18. Nat Biotechnol 14, 1675 (1996); 19. Cytometry 15, 267 (1994); 20. J Immunol Methods 138, 257 (1991); 21. Cytometry 12, 350 (1991); 22. J Immunol Methods 126, 69 (1990); 23. Cytometry 10, 426 (1989); 24. US 5,132,432; 25. Anal Biochem 198, 119 (1991); 26. US 5,830,912; 27. Flow Cytometry Sorting, 2nd Ed., Melamed MR, Lindmo T, Mendelsohn ML, Eds. pp. 367 380 (1990); 28. J Immunol Methods 219, 57 (1998); 29. J Histochem Cytochem 48, 1593 (2000). Custom Immunogen Preparation Low molecular weight molecules (<2000 daltons) or haptens generally will not elicit an immune response unless conjugated to a carrier protein such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Preparing these immunogens often requires introducing reactive groups into the haptens through chemical synthesis. Molecular Probes has considerable experience synthesizing reactive chemical species, including reactive forms of drugs, natural products and herbicides. In addition to their use for preparing immunogens, these reactive haptens can be used to generate new detection reagents and site-selective probes, as well as affinity matrices for isolating antibodies and receptors. We provide our custom services on an exclusive or nondisclosure basis when requested. Please contact our Custom and Bulk Sales Department for further information: In the U.S. and Canada: Phone: (541) 465-8390 Fax: (541) 984-5658 E-mail: custom@probes.com In Europe: Phone: +31-71-5236850 Fax: +31-71-5233419 E-mail: custom@probes.nl 196 Chapter 7 Antibodies, Avidins, Lectins and Related Products www.probes.com