6.4 Phycobiliproteins

Transcription

1 6.4 Phycobiliproteins Phycobiliproteins Phycobiliproteins are a family of reasonably stable and highly soluble fluorescent proteins derived from cyanobacteria and eukaryotic algae. These proteins contain covalently linked tetrapyrrole groups that play a biological role in collecting light and, through fluorescence resonance energy transfer, conveying it to a special pair of chlorophyll molecules located in the photosynthetic reaction center. 1 Because of their role in light collection, phycobiliproteins have evolved to maximize both absorption and fluorescence and to minimize the quenching caused either by internal energy transfer or by external factors such as changes in ph or ionic composition. 2,3 Phycobiliproteins have several advantages when used as fluorescent probes. These include: Intense long-wavelength excitation and emission to provide fluorescence that is relatively free of interference from other biological materials Relatively large Stokes shifts with extremely high emission quantum yields Fluorescence that is not quenched by external agents because the fluorophores are protected by covalent binding to the protein backbone Very high water solubility A homogeneous structure with defined molecular weights Multiple sites for stable conjugation to many biological and synthetic materials streptavidin, researchers have detected fewer than 100 receptorbound biotinylated antibodies per cell by flow cytometry. 13 A multistep amplification method utilizing a fluoresceinated opioid, biotinylated anti-fluorescein/oregon Green antibody (A-982, Section 7.4) and a phycoerythrin conjugate of avidin (A-2660) was required to detect low-abundance kappa opioid receptors by flow cytometry. Allophycocyanin and its conjugates are both brighter and more photostable than Cy5 conjugates (Figure 6.28), likely making APC the most sensitive fluorophore currently available for detection in laser-scanning microscopes that utilize the 633 nm output of the He Ne laser or the 647 nm spectral line of the Ar Kr laser, although our TSA kits that contain Alexa Fluor 647 tyramide (Section 6.2, Table 6.1) have the potential of yielding even greater signals by a horseradish peroxidase catalyzed method (Figure 6.6). B-PE is more photostable than R-PE but photostability of R-PE conjugates can be improved by adding 1-propyl gallate. 14 Spectral Characteristics of Phycobiliproteins B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin The phycobiliproteins B-phycoerythrin (B-PE), R-phycoerythrin (R-PE) and allophycocyanin (APC) are among the best dyes currently available for applications that require either high sensitivity or simultaneous multicolor detection. 4 6 Quantum yields up to 0.98 and extinction coefficients up to 2.4 million cm -1 M -1 have been reported for these fluorescent proteins (Table 6.2). On a molar basis, the fluorescence yield is equivalent to at least 30 unquenched fluorescein or 100 rhodamine molecules at comparable wavelengths. The fluorescence of a single molecule of B-PE has been detected. 7,8 In practical applications such as flow cytometry and immunoassays, 9,10 the sensitivity of B-PE and R-PE conjugated antibodies is usually five- to ten-times greater than that of the corresponding fluorescein conjugate. 11,12 Using R-PE conjugated 0 sec 30 sec 60 sec Figure 6.28 A comparison of the photobleaching rates of APC and Cy5 conjugates. The microtubules of bovine pulmonary artery endothelial (BPAE) cells were stained with mouse anti α-tubulin antibody (A-11126) in combination with goat anti mouse IgG labeled antibody with either crosslinked APC (A-865, 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. Table 6.2 Spectral data for B-PE, R-PE and APC. Cat # Phycobiliprotein Molecular Weight Absorption Max (nm) EC (cm -1 M -1 ) Emission Max (nm) Fluorescence QY P-800 B-phycoerythrin 240, , 565 2,410, P-801 R-phycoerythrin 240, , 546, 565 1,960, A-803, A-819 Allophycocyanin 104, , EC = Extinction coefficient. QY = Quantum yield. Section

2 Figure 6.29 Normalized absorption spectra for B-PE, R-PE and APC. Figure 6.30 Normalized fluorescence emission spectra for B-PE, R-PE and APC. Figure 6.31 Normalized fluorescence emission spectra of 1) Alexa Fluor 488 goat anti mouse IgG antibody (A-11001), 2) R-phycoerythrin goat anti mouse IgG antibody (P-852), 3) Alexa Fluor 610 Rphycoerythrin goat anti mouse IgG antibody (A-20980), 4) Alexa Fluor 647 R-phycoerythrin goat anti mouse IgG antibody (A-20990), and 5) Alexa Fluor 680 Rphycoerythrin goat anti mouse IgG antibody (A-20983). 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 6.29 and Figure 6.30 show the spectra for B-PE, R-PE and APC. R-PE can be excited efficiently at 488 nm either with an argon-ion laser or with a broadband illumination source (xenon- or mercury-arc lamps) and a standard fluorescein optical filter set (Table 24.8). With the proper emission filters, fluorescein (or any of the principal fluorescein substitutes described in Chapter 1) and R-PE can be simultaneously detected at approximately 520 nm and at wavelengths longer than 575 nm, respectively, making R-PE conjugates ideal for multicolor flow cytometry applications. One of the fluorescent microsphere suspensions in our CompenFlow Flow Cytometry Compensation Kit (C-7301, Section 24.2) has emission spectra that almost exactly match those of the phycoerythrins (Figure 24.28). This microsphere suspension is designed to help flow cytometry operators set up compensation circuits that properly remove unwanted phycoerythrin signals from secondary channels. Conjugates prepared from APC are ideal for use with the 633 nm spectral line of the red He Ne laser, 10,15 or the 647 nm spectral line of the krypton-ion laser. Tandem Conjugates of Phycobiliproteins A phycoerythrin-labeled detection reagent can be used in combination with a greenfluorescent detection reagent to detect two different signals using simultaneous excitation with the 488 nm spectral line of the argon-ion laser. 16 By conjugating R-PE to longerwavelength light emitting fluorescence acceptors, an energy transfer cascade is established wherein excitation of the R-PE produces fluorescence of the acceptor dye by the process of fluorescence resonance energy transfer (FRET, see Section 1.3). This process, which occurs naturally within single molecules and assemblies of phycobiliproteins (phycobilisomes), can be quite efficient, resulting in almost total transfer of energy from the phycobiliprotein to the acceptor dye of these tandem conjugates. Thus, it is possible to combine a green-fluorescent antibody conjugate with an R-PE conjugate, as mentioned above, and then to add tandem conjugates of R-PE with either our Alexa Fluor 610, Alexa Fluor 647 or Alexa Fluor 680 dyes for simultaneous detection of up to five targets using only 488 nm excitation (Figure 6.31, Figure 6.32, Figure 6.33). We have also conjugated APC to our Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes to provide tandem conjugates that can be excited by the He Ne laser at 633 nm or by the krypton-ion laser at 647 nm. These Alexa Fluor dye APC tandem conjugates can potentially be combined with direct APC conjugates for simultaneous three- or fourcolor applications (Figure 6.34). As the absorption and emission maxima of the acceptor dye move to longer wavelengths, the energy transfer efficiency from the R-PE to the bound dyes tends to decrease; also, the quantum yields of the longer-wavelength acceptor dyes in the conjugates tend to be lower than those of the shorter-wavelength dyes and to decrease further at high degrees of substitution. Consequently, the preparation of tandem conjugates necessarily involves careful optimization of both the energy transfer efficiency from the R-PE to the longer-wavelength emitting acceptor dye and the total brightness of the tandem conjugate (Figure 6.35, Figure 6.36). For our Alexa Fluor 647 and Alexa Fluor 680 tandem conjugates of R-PE, the energy transfer efficiency from R-PE to the attached dyes is about 99% and 98%, respectively, as determined from their fluorescence at 575 nm relative to unconjugated R-PE. The residual signal that overlaps the unquenched R-PE emission can be compensated by methods familiar to flow cytometrists. Phycoerythrin has previously been conjugated to our Texas Red dye to provide a third signal that is excitable at 488 nm; however, we feel that our Alexa Fluor 610 conjugates of R-phycoerythrin (A-20980, A-20981, S-20982) have emission properties superior to those of commercially available Texas Red conjugates of R-phycoerythrin. Not only are the Alexa Fluor 610 R-PE tandem conjugates more fluorescent than the commercially available Texas Red R-PE tandem conjugates, but also the fluorescence emission of Alexa Fluor 610 R-phycoerythrin tandem conjugates is shifted to somewhat longer wavelengths than is the emission of Texas Red R-PE conjugates, resulting in better separation from the emission of R-PE. Our Alexa Fluor 647 (A-20990, A-20991, S-20992) and Alexa Fluor 680 (A-20983, A-20984, S-20985) conjugates of R-PE have emission spectra (Figure 6.35) almost identical to those of Cy5 R-PE and Cy5.5 R-PE conjugates but tend to have more intense long-wavelength emission and to require less compensation in the R-PE channel than do the tandem conjugates of the Cy dyes (Figure 6.35, Figure 6.37). 168 Chapter 6 Ultrasensitive Detection Technology

3 Figure 6.34 Normalized fluorescence emission spectra of 1) allophycocyanin, crosslinked, goat anti mouse IgG antibody (A-865), 2) Alexa Fluor 680 allophycocyanin goat anti mouse IgG antibody (A-21000), 3) Alexa Fluor 700 allophycocyanin goat anti mouse IgG antibody (A-21003) and 4) Alexa Fluor 750 allophycocyanin goat anti mouse IgG antibody (A-21006). 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. Figure 6.32 Simultaneous detection of three cell surface markers using an Alexa Fluor 610 Rphycoerythrin tandem conjugate, Alexa Fluor 488 dye and R-phycoerythrin labels. Lymphocytes from ammonium chloride RBC lysed whole blood were labeled with a biotinylated mouse anti human CD3 monoclonal antibody (Caltag Laboratories), washed with 1% BSA in PBS and then incubated with Alexa Fluor 610 R-phycoerythrin tandem dye labeled streptavidin (S-20982). Cells were again washed and then labeled with directly conjugated primary antibodies against the CD8 and CD4 markers (Alexa Fluor 488 dye labeled mouse anti human CD8 antibody (A-21339) and R-phycoerythrin conjugated mouse anti human CD4 antibody (A-21337). After a further wash in 1% BSA/PBS, labeling was analyzed on a Becton Dickinson FACScan flow cytometer using excitation at 488 nm. CD8 was detected in the green channel (525 ± 10 nm), CD4 in the orange channel (575 ± 10 nm) and CD3 in the far red channel (>650 nm). The bivariate scatter plots show the expected mutually exclusive populations of CD4 and CD8 positive cells (panel A), together with co-positive CD3/CD4 (panel B) and CD3/CD8 (panel C) populations. Figure 6.33 Simultaneous detection of three cell surface markers using an Alexa Fluor 647 Rphycoerythrin tandem conjugate, Alexa Fluor 488 dye and R-phycoerythrin labels. Lymphocytes from ammonium chloride RBC lysed whole blood were labeled with a mouse anti human CD3 monoclonal antibody (Caltag Laboratories), washed with 1% BSA in PBS and then incubated with a goat anti mouse IgG antibody labeled with the Alexa Fluor 647 R-phycoerythrin tandem dye (A-20990). Cells were again washed and then labeled with directly conjugated primary antibodies against the CD8 and CD4 markers (Alexa Fluor 488 dye labeled mouse anti human CD8 (A-21339) and R-phycoerythrin conjugated mouse anti human CD4 antibody (A-21337). After a further wash in 1% BSA/PBS, labeling was analyzed on a Becton Dickinson FACScan flow cytometer using excitation at 488 nm. CD8 was detected in the green channel (525 ± 10 nm), CD4 in the orange channel (575 ± 10 nm) and CD3 in the far-red channel (>650 nm). The bivariate scatter plots show the expected mutually exclusive populations of CD4 and CD8 positive cells (panel A), together with co-positive CD3/CD4 (panel B) and CD3/CD8 (panel C) populations. Figure 6.35 Fluorescence emission spectra of Alexa Fluor 647 R-phycoerythrin streptavidin (S-20992; 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. Section

4 Pure Phycobiliproteins Molecular Probes was the first company to make the phycobiliproteins available for research and we can supply bulk quantities of B-PE (P-800), R-PE (P-801), APC (A-803), chemically crosslinked APC (A-819) or their conjugates at a considerable discount. Phycobiliproteins may undergo some loss of fluorescence upon freezing. The pure proteins are shipped in ammonium sulfate suspension and are stable for at least one year when stored at 4 C. The conjugates and modified derivatives are shipped in solutions containing sodium azide to inhibit bacterial growth and typically have a useful life of more than six months. All phycobiliproteins and their derivatives should be stored refrigerated, never frozen. Phycobiliprotein Conjugates Reactive Phycobiliprotein Derivative Conjugates of R-phycoerythrin with other proteins are generally prepared from the pyridyldisulfide derivative of R-PE (P-806). This derivative can be directly reacted with thiolated antibodies, enzymes and other biomolecules to form a disulfide linkage. More commonly, the pyridyldisulfide groups in this derivative are first reduced to thiols, which are then reacted with maleimide- or iodoacetamide-derivatized proteins (Figure 5.3). Figure 6.36 Fluorescence emission spectra of Alexa Fluor 610 R-phycoerythrin streptavidin (S-20982; 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 6.38 Analytical size-exclusion chromatograms of free streptavidin (S-888; red curve, detected by absorption at 280 nm) and R-phycoerythrin streptavidin (S-866; blue curve, detected by absorption at 565 nm), demonstrating that the R- phycoerythrin conjugate is substantially free of unconjugated streptavidin. Figure 6.37 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 (S-20992) 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 emission bandpass filter (675 ± 20 nm; upper panels) or a longpass filter (>650 nm; lower panels). In each histogram, unstained and stained cells are represented by blue and red lines, respectively. The numbers above each peak represent mean channel fluorescence intensities. Data provided by William Telford, NCI-NIH, Bethesda, MD. 170 Chapter 6 Ultrasensitive Detection Technology

5 Because the pyridyldisulfide derivative of R-PE is somewhat unstable, we recommend using it within three months of receipt. Phycobiliproteins can be conveniently crosslinked to other proteins using the reagents and protocol provided in our Protein Protein Crosslinking Kit (P-6305, Section 5.2). Phycobiliprotein-Labeled Secondary Detection Reagents Molecular Probes regularly prepares R-PE conjugates of the goat anti mouse IgG (P-852) and goat anti rabbit IgG (P-2771) antibodies and NeutrAvidin biotin-binding protein (A-2660), as well as the R-PE conjugate of streptavidin (S-866). R-PE conjugates of anti mouse IgG, IgG 2a, IgG 2b and IgG 3 antibodies are also available (P-21129, P-21139, P-21149, P-21159). Our streptavidin conjugate of R-PE has been purified to ensure that all unconjugated streptavidin has been removed (Figure 6.38), making it especially suitable for detecting biotinylated probes on microarrays (Section 8.5, Figure 6.39). We also prepare a special tetramer grade of streptavidin R-PE (S-21388) that is a further fractionation of our product S-866 (see MHC Tetramer Technology). Because allophycocyanin tends to dissociate into subunits when highly diluted or treated with chaotropic agents, Molecular Probes prepares its APC conjugates of the goat anti mouse IgG (A-865), rabbit anti mouse IgG (A-10930) and goat anti rabbit IgG (A-10931) antibodies and of streptavidin (S-868) from chemically crosslinked APC (A-819), a protein complex that does not dissociate, even in strongly chaotropic salts In addition, biotinylated R-PE (P-811) can be used with standard avidin/streptavidin bridging techniques to detect biotinylated molecules. 26 Tandem Conjugates of Phycobiliproteins We have conjugated R-phycoerythrin (R-PE) with three of our Alexa Fluor dyes the Alexa Fluor 610, Alexa Fluor 647 and Alexa Fluor 680 dyes then conjugated these fluorescent proteins to antibodies or streptavidin to yield tandem conjugates that can be excited with the 488 nm spectral line of the argon-ion laser (Table 6.3). The long-wavelength emission maxima are 627 nm for the Alexa Fluor 610 R-PE conjugates, 667 nm for the Alexa Fluor 647 R-PE conjugates and 702 nm for the Alexa Fluor 680 R-PE conjugates (Figure 6.31). Emission of the Alexa Fluor 610 R-PE conjugates is shifted to longer wavelengths by about 13 nm relative to that of Texas Red conjugates of R-PE (Figure 6.36). The Alexa Fluor 647 R-PE tandem conjugates have spectra virtually identical to those of Cy5 conjugates of R-PE but are about three-fold brighter (Figure 6.35). These tandem conjugates can potentially be used for simultaneous three-, four- or five-color labeling with a single excitation (Figure 6.31, Figure 6.32, Figure 6.33). In addition, we have conjugated APC to our Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes to provide tandem conjugates that can be excited by the He Ne laser at 633 nm or by the krypton-ion laser at 647 nm with emission beyond 700 nm (Table 6.4). Our Alexa Fluor dye APC tandem conjugates can potentially be combined with direct APC conjugates for simultaneous three-color applications (Figure 6.34). Figure 6.39 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 (S-866). 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); nm excitation (He Ne laser)/570 nm emission filter (middle); nm excitation (He Ne laser)/ 592 nm emission filter (right). Table 6.3 Tandem conjugates of R-phycoerythrin (R-PE). Acceptor Dye (Ex/Em) * Alexa Fluor 610 (565/627) Alexa Fluor 647 (565/667) Alexa Fluor 680 (565/702) Anti Mouse IgG Conjugate Anti Rabbit IgG Streptavidin A A S A A S A A S * Fluorescence excitation and emission maxima, in nm. Host = goat. Table 6.4 Tandem conjugates of allophycocyanin (APC). Acceptor Dye (Ex/Em) * Alexa Fluor 680 (650/702) Alexa Fluor 700 (650/719) Alexa Fluor 750 (650/779) Anti Mouse IgG Conjugate Anti Rabbit IgG Streptavidin A A S A A S A A S * Fluorescence excitation and emission maxima, in nm. Host = goat. Section

6 Zenon One Technology Chemical conjugation of phycobiliproteins to antibodies and other proteins is a moderately difficult and relatively low-yield process that cannot be done on very small quantities of proteins. Our Protein Protein Crosslinking Kit (P-6305, Section 5.2) provides the reagents and a protocol for conjugations of phycobiliproteins using our recommended procedure; however, researchers have typically used secondary antibodies to label their mouse monoclonal primary antibodies. Unfortunately, normally only one secondary anti mouse IgG antibody can be used per experiment, thus necessitating access to directly conjugated primary antibodies, use of primary antibodies from unrelated species of animals or combinations of biotin avidin reagents with directly labeled primary and secondary antibodies for most multicolor experiments in both imaging and flow cytometry. Although suitable for multicolor experiments, directly labeled primary antibodies may not be commercially available or are more expensive than unlabeled antibodies. Also, a whole arsenal of direct conjugates may be required for some immunofluorescence applications. Our Zenon One mouse IgG 1 labeling technology, which is described in detail in Section 7.2, makes it possible to rapidly form stable complexes with the Fc fragment of mouse and rat IgG 1 antibodies. The Zenon One procedure (Figure 7.32) has numerous advantages, particularly when using phycobiliproteinconjugated reagents: Conjugations can be done on submicrograms of a primary antibody. The reactions are usually quantitative with respect to the primary antibody. TECHNICAL NOTE MHC Tetramer Technology Cytotoxic T lymphocytes (CTLs) or killer T cells perform a crucial function in the vertebrate immune system they serve to destroy cells that display foreign antigens on their surfaces. CTLs are distinguished from other types of T lymphocytes (such as helper T cells) by their expression of CD8, a transmembrane protein with immunoglobulin-like domains that interact with class I Major Histocompatibility Complexes (MHCs). 1 MHC molecules act in a dual role; their amino-terminal domains bind antigen for presentation to T cells, as well as serving as a marker identifying host cells to the immune system. Activation of CTLs to eliminate a target cell occurs only if the CD8 protein recognizes the proper MHC molecule and the α/β T-cell receptor binds the MHC-presented antigen. 2 Historically, assaying for an antigen-specific T-cell response made use of the limiting dilution analysis (LDA) method, which requires that single clones survive, divide and differentiate before they can be detected. However, this method likely underestimates the number of CTLs that respond to a particular antigen because it will not detect cells that are part of the expanded effector population that can no longer divide. 3 Alternate approaches attempted to directly measure CTL response using an antigenic peptide and MHC. However, these experiments were hampered by the low affinity of the interaction between the MHC/antigen complex and the corresponding CTL receptors. 4 Joining multiple copies of the MHC/antigen complex into a single probe tetramer technology resolved the difficulties presented by the low affinity of the class I MHC molecule for the CD8 receptor. 5 CTL-response assays using tetramer technology often detect a response rate that is times higher than detected by the LDA method, 3 and have proven indispensable for the study of the CTL response to HIV, 6 cancers 7 and transplants. 8 Similar methods using class II MHC tetramers are also being employed to explore the response of helper (CD-4 expressing) T cells. 9 MHC tetramers are formed by first refolding MHCs in the presence of high concentrations of the desired antigenic peptide, followed by biotinylation of the carboxy-terminus of one chain of the MHC molecule. This MHC/peptide complex can then be bound to streptavidin. Because the latter has four biotin-binding sites, four An MHC tetramer. MHC molecules can be linked together in a single complex. The use of fluorophore-labeled streptavidin or streptavidin-coated magnetic beads for tetramer formation allows for efficient detection by flow cytometry or immunomagnetic methods. 10 It is crucial for MHC tetramer based assays that the labeled streptavidin be free of unlabeled protein because this lowers the apparent activity of the MHC complex by blocking the biotinylation site from binding to the labeled streptavidin. Molecular Probes special tetramer grade streptavidin conjugate of R-PE (S-21388) has been purified to ensure that all unconjugated streptavidin has been removed, making it especially suitable for tetramer technology. References 1. Annu Rev Immunol 10, 645 (1992); 2. Adv Immunol 53, 59 (1993); 3. J Exp Med 187, 1367 (1998); 4. Proc Natl Acad Sci U S A 91, (1994); 5. Immunol Rev 150, 5 (1996); 6. Science 279, 2103 (1998); 7. J Immunol 162, 2227 (1999); 8. Nat Med 5, 839 (1999); 9. J Clin Invest 104, 1669 (1999); 10. Nat Med 6, 707 (2000). 172 Chapter 6 Ultrasensitive Detection Technology

7 Labeling and purification of the complex can be completed in only minutes. Labeling is essentially irreversible under conditions of use. Multiple mouse (or rat) IgG 1 antibodies can be used in the same experiment. Some crossreactivity of the Zenon One reagents with other mouse isotypes may be observed. Antibody complexes prepared from the Zenon One Labeling Kits can be combined with direct conjugates for multicolor labeling. Zenon One labeling is possible with almost any of Molecular Probes dyes including R-PE (Z-25055) and APC (Z-25051) as well as most of our Alexa Fluor dyes (Table 7.1). Zenon One Labeling Kits with tandem conjugates of phycobiliproteins are under development at Molecular Probes and these conjugates should permit further combinations of detection wavelengths to be used. Zenon One labeling of mouse IgG 1 antibodies with horseradish peroxidase and alkaline phosphatase is also practical (Section 7.2) and can be used in combination with our TSA and ELF technologies (Section 6.2, Section 6.3). Zenon One labeling of other mouse and rat isotypes and antibodies from other species with the Zenon One reagents tends to be less efficient; however, similar Zenon labeling kits are under development. Anti-CD3, Anti-CD4 and Anti-CD8 Antibodies Molecular Probes now offers three mouse monoclonal antihuman T-cell markers, anti-cd3, anti-cd4 and anti-cd8 antibodies. These antibodies are available unlabeled or conjugated to one of our superior Alexa Fluor dyes (Table 7.15) or to R-phycoerythrin (A-21333, A-21337, A-21341). The approximate absorption and fluorescence emission maxima for each of the conjugates are shown in Table Research applications for the anti-cd antibodies include: the identification and enumeration of CD3 +, CD4 + and CD8 + cells by flow cytometry (Figure 6.32, Figure 6.33); the visualization of CD + cells by immunohistochemistry in acetone-fixed, frozentissue sections; the immunoprecipitation of CD + fractions; and the isolation or removal of T cells by cell sorting or bio-panning. 27 Anti-CD3 antibodies help in the identification of T cells. CD3, a member of the immunoglobulin superfamily, is a cluster of differentiation (CD) cell-surface antigen associated with the T-cell receptor (TcR) of T cells and thymocytes. TcRs are specific for complexes comprising short peptides bound to and presented by the major histocompatibility complex (MHC). The human CD3/TcR complex is made up of at least five CD3 proteins (γ,, ε, η, ζ) in association with either alpha/beta or gamma/delta proteins of the TcR. 28,29 The TcR recognizes and binds to antigens presented by the MHC, after which the protein chains of the CD3 complex mediate activation signals. The CD3 molecule is not found on B cells and, thus, can be used as a marker for T cells. Anti-CD4 and anti-cd8 antibodies can be used to differentiate between helper T cells and killer T cells, both of which are CD3 +. Helper/inducer T cells express the cell-surface CD4 antigen, which interacts with MHC class II molecules and is the primary receptor for the human immunodeficiency virus (HIV). 30 Killer T cells express the CD8 cell-surface antigen, which interacts with MHC class I molecules. The interaction results in the activation of the killer T cell and an increase in its avidity for the corresponding target cells. R-Phycoerythrin Anti-Fluorescein/Oregon Green Antibody Our R-phycoerythrin conjugate of the rabbit anti-fluorescein/ Oregon Green antibody (A-21250) has the unique utility of both shifting the green-fluorescence emission of fluorescein-labeled probes to longer wavelengths and greatly intensifying the signal. Anti-fluorescein antibodies strongly crossreact with our Oregon Green dye conjugates, suggesting the possibility of amplifying the signal from nucleic acid probes labeled by our ARES Oregon Green 488 DNA Labeling Kit (A-21674, Section 8.2) or ULYSIS Oregon Green 488 Nucleic Acid Labeling Kit (U-21659, Section 8.2) or for further amplifying the signal of Oregon Green 488 tyramide, which is used in three of our tyramide signal amplification (TSA) Kits (Section 6.2, Table 6.1). Custom Phycobiliprotein Conjugates Molecular Probes has carried out hundreds of successful conjugations with phycobiliproteins, beginning soon after their use was disclosed in We are experts in doing custom conjugations of phycobiliproteins to antibodies and other proteins and welcome inquiries for specific conjugates. For more information or a quote, please contact Molecular Probes Custom and Bulk Sales Department. References 1. J Fluorescence 1, 135 (1991); 2. J Biol Chem 264, 1 (1989); 3. Methods Enzymol 167, 291 (1988); 4. Trends Biochem Sci 9, 423 (1984); 5. Proc Natl Acad Sci U S A 85, 7312 (1988); 6. J Cell Biol 93, 981 (1982); 7. Proc Natl Acad Sci U S A 86, 4087 (1989); 8. Anal Chem 59, 2158 (1987); 9. J Histochem Cytochem 39, 921 (1991); 10. Anal Lett 24, 1075 (1991); 11. Eur Biophys J 15, 141 (1987); 12. Clin Chem 29, 1582 (1983); 13. J Immunol Methods 135, 247 (1990); 14. Anal Biochem 161, 442 (1987); 15. Cytometry 15, 267 (1994); 16. J Immunol Methods 243, 77 (2000); 17. Biotechniques 31, 490 (2001); 18. Proc Natl Acad Sci U S A 98, 8862 (2001); 19. Proc Natl Acad Sci U S A 97, 2680 (2000); 20. Proc Natl Acad Sci U S A 97, 3260 (2000); 21. Proc Natl Acad Sci U S A 95, 3752 (1998); 22. Nat Biotechnol 14, 1675 (1996); 23. Arch Biochem Biophys 223, 24 (1983); 24. Biochemistry 19, 2817 (1980); 25. Cytometry 8, 91 (1987); 26. J Biol Chem 265, (1990); 27. Immunobiology 203, 769 (2001); 28. Leucocyte Typing IV: White Cell Differentiation Antigens, Knapp, W, et al., Ed. pp (1989); 29. Annu Rev Immunol 6, 629 (1988); 30. Science 271, 173 (1996). Section

8 Product List 6.4 Phycobiliproteins Cat # Product Name Unit Size A Alexa Fluor 680 allophycocyanin goat anti-mouse IgG (H+L) *1 mg/ml* µl A Alexa Fluor 700 allophycocyanin goat anti-mouse IgG (H+L) *1 mg/ml* µl A Alexa Fluor 750 allophycocyanin goat anti-mouse IgG (H+L) *1 mg/ml* µl A Alexa Fluor 680 allophycocyanin goat anti-rabbit IgG (H+L) *1 mg/ml* µl A Alexa Fluor 700 allophycocyanin goat anti-rabbit IgG (H+L) *1 mg/ml* µl A Alexa Fluor 750 allophycocyanin goat anti-rabbit IgG (H+L) *1 mg/ml* µl A Alexa Fluor 610 R-phycoerythrin goat anti-mouse IgG (H+L) *1 mg/ml* µl A Alexa Fluor 647 R-phycoerythrin goat anti-mouse IgG (H+L) *1 mg/ml* µl A Alexa Fluor 680 R-phycoerythrin goat anti-mouse IgG (H+L) *1 mg/ml* µl A Alexa Fluor 610 R-phycoerythrin goat anti-rabbit IgG (H+L) *1 mg/ml* µl A Alexa Fluor 647 R-phycoerythrin goat anti-rabbit IgG (H+L) *1 mg/ml* µl A Alexa Fluor 680 R-phycoerythrin goat anti-rabbit IgG (H+L) *1 mg/ml* µl A-803 allophycocyanin *4 mg/ml* ml A-819 allophycocyanin, crosslinked (APC-XL) *4 mg/ml* µl A-865 allophycocyanin, crosslinked, goat anti-mouse IgG (H+L) *1 mg/ml* ml A allophycocyanin, crosslinked, goat anti-rabbit IgG (H+L) *1 mg/ml* ml A allophycocyanin, crosslinked, rabbit anti-mouse IgG (H+L) *1 mg/ml* ml A anti-cd3, mouse IgG 1, monoclonal , R-phycoerythrin conjugate *0.2 mg/ml* ml A anti-cd4, mouse IgG 1, monoclonal , R-phycoerythrin conjugate *0.2 mg/ml* ml A anti-cd8, mouse IgG 2a, monoclonal , R-phycoerythrin conjugate *0.2 mg/ml* ml A anti-fluorescein/oregon Green, rabbit IgG fraction, R-phycoerythrin conjugate *2 mg/ml* µl A-2660 avidin, NeutrAvidin, R-phycoerythrin conjugate *1 mg/ml*... 1 ml P-800 B-phycoerythrin *4 mg/ml* ml P-801 R-phycoerythrin *4 mg/ml* ml P-811 R-phycoerythrin, biotin-xx conjugate *4 mg/ml* ml P R-phycoerythrin goat anti-mouse IgG 1 (γ1) conjugate *1 mg/ml* µl P R-phycoerythrin goat anti-mouse IgG 2a (γ2a) conjugate *1 mg/ml* µl P R-phycoerythrin goat anti-mouse IgG 2b (γ2b) conjugate *1 mg/ml* µl P R-phycoerythrin goat anti-mouse IgG 3 (γ3) conjugate *1 mg/ml* µl P-852 R-phycoerythrin goat anti-mouse IgG (H+L) *1 mg/ml*... 1 ml P-2771 R-phycoerythrin goat anti-rabbit IgG (H+L) *1 mg/ml* ml P-806 R-phycoerythrin, pyridyldisulfide derivative *2 mg/ml*... 1 ml S streptavidin, Alexa Fluor 680 allophycocyanin conjugate (Alexa Fluor 680 allophycocyanin streptavidin) S streptavidin, Alexa Fluor 700 allophycocyanin conjugate (Alexa Fluor 700 allophycocyanin streptavidin) S streptavidin, Alexa Fluor 750 allophycocyanin conjugate (Alexa Fluor 750 allophycocyanin streptavidin) S streptavidin, Alexa Fluor 610 R-phycoerythrin conjugate (Alexa Fluor 610 R-phycoerythrin streptavidin) S streptavidin, Alexa Fluor 647 R-phycoerythrin conjugate (Alexa Fluor 647 R-phycoerythrin streptavidin) S streptavidin, Alexa Fluor 680 R-phycoerythrin conjugate (Alexa Fluor 680 R-phycoerythrin streptavidin) S-868 streptavidin, allophycocyanin, crosslinked, conjugate *1 mg/ml* ml S-866 streptavidin, R-phycoerythrin conjugate *1 mg/ml*... 1 ml S streptavidin, R-phycoerythrin conjugate *tetramer grade**1 mg/ml*... 1 ml Z Zenon One Alexa Fluor 700 allophycocyanin Mouse IgG 1 Labeling Kit... 1 kit Z Zenon One Alexa Fluor 750 allophycocyanin Mouse IgG 1 Labeling Kit... 1 kit Z Zenon One Alexa Fluor 610 R-phycoerythrin Mouse IgG 1 Labeling Kit... 1 kit Z Zenon One Alexa Fluor 647 R-phycoerythrin Mouse IgG 1 Labeling Kit... 1 kit Z Zenon One Alexa Fluor 680 R-phycoerythrin Mouse IgG 1 Labeling Kit... 1 kit Z Zenon One Allophycocyanin Mouse IgG 1 Labeling Kit *50 labelings*... 1 kit Z Zenon One R-phycoerythrin Mouse IgG 1 Labeling Kit *50 labelings*... 1 kit 174 Chapter 6 Ultrasensitive Detection Technology