Figure 6.4.4 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.
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Phycobiliproteins are a family of highly soluble and reasonably stable 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.
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. Phycobiliproteins have several advantages when used as fluorescent probes, including:
- 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
- Homogeneous structure with defined molecular weights
- Multiple sites for stable conjugation to many biological and synthetic materials
B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin
The phycobiliproteins B-phycoerythrin (B-PE), R-phycoerythrin (R-PE) and allophycocyanin (APC) are among the preferred dyes for applications that require either high sensitivity or simultaneous multicolor detection. Quantum yields up to 0.98 and extinction coefficients up to 2.4 million cm-1M-1 have been reported for these fluorescent proteins (Spectral data for B-PE, R-PE and APC—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. B-PE is reportedly more photostable than R-PE, but photostability of R-PE conjugates can be improved by adding 1-propyl gallate.
In practical applications such as flow cytometry and immunoassays, the sensitivity of B-PE– and R-PE–conjugated antibodies is usually 5 to 10 times greater than that of the corresponding fluorescein conjugate. Using R-PE–conjugated streptavidin, researchers have detected fewer than 100 receptor-bound biotinylated antibodies per cell by flow cytometry. A multistep amplification method utilizing a fluoresceinated opioid, biotinylated anti–fluorescein/Oregon Green dye antibody (A982, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) and a phycoerythrin conjugate of avidin (A2660) was required to detect low-abundance κ-opioid receptors by flow cytometry. In imaging applications, APC and its conjugates are both brighter and more photostable than Cy5 conjugates (Figure 6.4.1).
Figure 6.4.2 and Figure 6.4.3 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 . With the proper emission filters, fluorescein (or any of the principal fluorescein substitutes described in Fluorophores and Their Amine-Reactive Derivatives—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.
Figure 6.4.1 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 6.4.2 Normalized absorption spectra for B-PE, R-PE and APC.
Figure 6.4.3 Normalized emission spectra for B-PE, R-PE and APC.
Tandem Conjugates of Phycobiliproteins
A phycoerythrin-labeled detection reagent can be used in combination with a green-fluorescent detection reagent to detect two different signals using simultaneous excitation with the 488 nm spectral line of the argon-ion laser. By conjugating R-PE to longer-wavelength 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) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). 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, Alexa Fluor 680 or Alexa Fluor 750 dyes for simultaneous detection of up to five targets using only 488 nm excitation (Figure 6.4.4).
Phycoerythrin has previously been conjugated to our Texas Red dye to provide a third signal that is excitable at 488 nm; however, our Alexa Fluor 610 tandem conjugates of R-PE (A20980, A20981, S20982) have emission properties superior to those of commercially available Texas Red tandem conjugates of R-PE. 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-PE 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 (Figure 6.4.5). Our Alexa Fluor 647 (A20990, A20991, S20992) and Alexa Fluor 680 (A20983, A20984, S20985) tandem conjugates of R-PE have emission spectra almost identical to those of Cy5–R-PE and Cy5.5–R-PE tandem conjugates but tend to have more intense long-wavelength emission and to require less compensation in the R-PE channel than the Cy dye–R-PE tandem conjugates (Figure 6.4.6, Figure 6.4.7). Our Alexa Fluor 750 tandem conjugate of R-PE (S32363) emits at 771 nm.
We have also conjugated APC to our Alexa Fluor 680 (A21000, A21001MP, S21002), Alexa Fluor 700 (A21005) and Alexa Fluor 750 (A21006, S21008) dyes to provide tandem conjugates that can be excited by the He-Ne laser at 633 nm. These Alexa Fluor dye–APC tandem conjugates can potentially be combined with direct APC conjugates for simultaneous three- or four-color applications (Figure 6.4.8).
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 tandem 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. For our Alexa Fluor 647 and Alexa Fluor 680 tandem conjugates of R-PE, the energy transfer efficiency from R-PE to the attached dye 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.
Figure 6.4.5 Fluorescence emission spectra of Alexa Fluor 610–R-phycoerythrin streptavidin (S20982; red) and Texas Red–R-phycoerythrin streptavidin (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 6.4.6 Fluorescence emission spectra of Alexa Fluor 647–R-phycoerythrin streptavidin (S20992; red) and Cy5–R-phycoerythrin streptavidin (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 6.4.7 Comparison of immunofluorescent staining by R-phycoerythrin–dye tandem conjugates. EL4 cells labeled with a biotinylated anti-CD44 monoclonal antibody 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 6.4.8 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
We were the first company to make the phycobiliproteins available for research, and we can supply bulk quantities of B-PE (P800), R-PE (P801), APC (A803) and chemically crosslinked APC (A819) ; please contact Invitrogen Custom Services for more information.
Phycobiliproteins may undergo some loss of fluorescence upon freezing. The pure proteins are shipped in an 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.
Reactive Phycobiliprotein Derivative
Conjugates of R-PE with other proteins are generally prepared from the pyridyldisulfide derivative of R-PE (P806). This derivative can be directly reacted with thiolated antibodies, enzymes and other biomolecules to form a disulfide linkage. More commonly, however, the pyridyldisulfide groups in this derivative are first reduced to thiols, which are then reacted with maleimide-derivatized proteins (Figure 6.4.9). 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 (P6305, Chemical Crosslinking Reagents—Section 5.2).
Figure 6.4.9 SPDP derivatization reactions. SPDP (S1531) reacts with an amine-containing biomolecule at pH 7 to 9, yielding a pyridyldithiopropionyl mixed disulfide. The mixed disulfide can then be reacted with a reducing agent such as DTT (D1532) or TCEP (T2556) to yield a 3-mercaptopropionyl conjugate or with a thiol-containing biomolecule to form a disulfide-linked tandem conjugate. Either reaction can be quantitated by measuring the amount of 2-pyridinethione chromophore released during the reaction.
Phycobiliprotein-Labeled Secondary Detection Reagents
We prepare R-PE conjugates of the goat anti–mouse IgG (P852) and goat anti–rabbit IgG (P2771MP) antibodies and NeutrAvidin biotin-binding protein (A2660), as well as both the R-PE (SAPE, S866) and B-PE (S32350) conjugates of streptavidin. R-PE conjugates of anti–mouse IgG1, IgG2a and IgG2b antibodies are also available (P21129, P21139, P21149). Our streptavidin conjugates of R-PE and B-PE have been purified to ensure that all unconjugated streptavidin has been removed (Figure 6.4.10), making them useful for multicolor flow cytometry and microarray assays (Figure 6.4.11). In addition, biotinylated R-PE (P811) can be used with standard avidin/streptavidin bridging techniques to detect biotinylated molecules.
Because APC tends to dissociate into subunits when highly diluted or treated with chaotropic agents, we prepare all APC conjugates—including APC tandem conjugates, APC conjugates of the goat anti–mouse IgG (A865) and goat anti–rabbit IgG (A10931) antibodies and APC-labeled streptavidin (S868)—from chemically crosslinked APC (A819), a protein complex that does not dissociate even in strongly chaotropic salts. We also prepare premium-grade R-PE and APC conjugates of streptavidin (S21388, S32362), which represent an even further fractionation of our R-PE streptavidin (S866) and APC streptavidin (S868), respectively.
Figure 6.4.10 Analytical size-exclusion chromatograms of free streptavidin (S888; red curve, detected by absorption at 280 nm) and R-phycoerythrin streptavidin (SAPE; S866, S21388; blue curve, detected by absorption at 565 nm), demonstrating that the R-phycoerythrin conjugate is substantially free of unconjugated streptavidin.
Figure 6.4.11 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).
Secondary Detection Reagents Labeled with Alexa Fluor Dye–Phycobiliprotein Tandem Conjugates
We have conjugated R-PE with four of our Alexa Fluor dyes—Alexa Fluor 610, Alexa Fluor 647, Alexa Fluor 680 and Alexa Fluor 750 dyes—and then conjugated these fluorescent proteins to antibodies or streptavidin to yield secondary detection reagents that can be excited with the 488 nm spectral line of the argon-ion laser (Tandem conjugates of R-phycoerythrin (R-PE)—Table 6.3). The long-wavelength emission maxima are 628 nm for the Alexa Fluor 610–R-PE conjugates, 668 nm for the Alexa Fluor 647–R-PE conjugates, 702 nm for the Alexa Fluor 680–R-PE conjugates (Figure 6.4.4) and 771 nm for the Alexa Fluor 750–R-PE conjugates. 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 6.4.5). 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 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 6.4.6). These tandem conjugates can potentially be used for simultaneous three-, four- or five-color labeling with a single excitation (Figure 6.4.4, Figure 6.4.12, Figure 6.4.13).
In addition, we have conjugated crosslinked APC (A819) to our Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes, and then conjugated these fluorescent proteins to antibodies or streptavidin to yield secondary detection reagents that can be excited with the He-Ne laser at 633 nm with emission beyond 700 nm (Tandem conjugates of allophycocyanin (APC)—Table 6.4). The long-wavelength emission maxima are 702 nm for the Alexa Fluor 680–APC conjugates, 719 nm for the Alexa Fluor 700–APC conjugates and 779 nm for the Alexa Fluor 750–APC conjugates (Figure 6.4.8). Our Alexa Fluor dye–APC tandem conjugates can potentially be combined with direct APC conjugates for simultaneous three-color applications (Figure 6.4.8).
Figure 6.4.12 Simultaneous detection of three cell surface markers using an Alexa Fluor 610–R-phycoerythrin 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, washed with 1% BSA in PBS and then incubated with Alexa Fluor 610–R-phycoerythrin tandem dye–labeled streptavidin (S20982). 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 and R-phycoerythrin–conjugated mouse anti–human CD4 antibody). 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 red channel (>650 nm). The bivariate scatter plots show the expected mutually exclusive populations of CD4 and CD8 positive cells (A), together with co-positive CD3/CD4 (B) and CD3/CD8 (C) populations.
Figure 6.4.13 Simultaneous detection of three cell surface markers using an Alexa Fluor 647–R-phycoerythrin 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, 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 (A20990). 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 and R-phycoerythrin–conjugated mouse anti–human CD4 antibody). 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 red channel (>650 nm). The bivariate scatter plots show the expected mutually exclusive populations of CD4 and CD8 positive cells (A), together with co-positive CD3/CD4 (B) and CD3/CD8 (C) populations.
R-Phycoerythrin Anti–Fluorescein/Oregon Green Dye Antibody
The R-PE conjugate of the rabbit anti–fluorescein/Oregon Green dye antibody (A21250) has the unique ability both to shift the green-fluorescent emission of fluorescein-labeled probes to longer wavelengths and to greatly intensify the signal (Figure 6.4.14). 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 ULYSIS Oregon Green 488 Nucleic Acid Labeling Kit (U21659, Labeling Oligonucleotides and Nucleic Acids—Section 8.2) or for further amplifying the signal of Oregon Green 488 tyramide, which is used in some of our TSA Kits (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2, Tyramide Signal Amplification (TSA) Kits—Table 6.1).
Figure 6.4.14 Color-shifting using a labeled anti–fluorescein/Oregon Green dye antibody. Jurkat cells were first stained with a primary mouse anti–human CD3 antibody, followed by fluorescein goat anti–mouse IgG antibody (F2761), with the resultant fluorescence detected in the R-phycoerythrin (red-orange fluorescence) channel of a flow cytometer (blue curve). The weak signal was then shifted to better suit the R-phycoerythrin channel by the addition of an R-phycoerythrin conjugate of anti–fluorescein/Oregon Green dye antibody (A21250). The resulting signal intensity is approximately two orders of magnitude greater (red curve) than the direct fluorescence from the first staining step (blue curve).
Phycobiliprotein Conjugates of Annexin V
In collaboration with Nexins Research BV, we offer the highly fluorescent APC and R-PE conjugates of annexin V (A35110, A35111), in addition to several other fluorescent annexin V conjugates (Assays for Apoptosis—Section 15.5). Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, an indicator of intermediate stages of apoptosis (Assays for Apoptosis—Section 15.5). Several of Molecular Probes apoptosis assay kits (V35112, V35113, V35114; Assays for Apoptosis—Section 15.5; Molecular Probes apoptosis assay kits—Table 15.4) contain either R-PE–annexin V or APC–annexin V conjugates as well as SYTOX Green nucleic acid stain to characterize mixed populations of apoptotic and nonapoptotic cells by flow cytometry.
Custom Phycobiliprotein Conjugates
We have carried out hundreds of successful conjugations with phycobiliproteins, beginning soon after their use was disclosed in 1982. We are experts in doing custom conjugations of phycobiliproteins to antibodies and other proteins and welcome inquiries for specific conjugates (Invitrogen Custom Services).
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. The Protein–Protein Crosslinking Kit (P6305, Chemical Crosslinking Reagents—Section 5.2) provides the reagents and a protocol for conjugating phycobiliproteins using our recommended procedure. However, instead of labeling each primary antibody, researchers typically use labeled secondary antibodies to detect their primary antibodies. Our exceptional Zenon immunolabeling technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) provides an easy, versatile and unique method of labeling antibodies with phycobiliproteins, as well as with many other premier dyes, haptens and enzymes. This enabling technology not only eliminates the need for secondary detection reagents in many applications, but also simplifies immunolabeling applications that were previously time consuming or impractical, including the use of multiple antibodies derived from the same species in the same protocol, as well as the detection of antibody binding in tissues when both the antibody and the tissue are derived from the same species.
Zenon immunolabeling technology allows the rapid and quantitative preparation of antibody complexes from a purified antibody fraction or from a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant. The Zenon antibody labeling procedure (Figure 6.4.15) has numerous advantages, particularly when preparing phycobiliprotein-labeled antibodies:
- Conjugations can be done on submicrogram quantities of a primary antibody.
- The reactions are usually quantitative with respect to the primary antibody.
- Labeling and purification of the complex can be completed in only minutes.
- Labeling is essentially irreversible under conditions of use.
- Multiple antibodies derived from the same species can be used in the same experiment.
- The fluorescence intensity of the cells can be adjusted by changing the ratio of labeling reagent to primary antibody, which even permits using identical dyes to detect multiple targets in cells by flow cytometry.
- Antibody complexes prepared from the Zenon Antibody Labeling Kits can be combined with direct conjugates for multicolor labeling.
- Labeling is possible with a wide variety of fluorophores, including R-PE and APC as well as most of our Alexa Fluor dyes (Zenon Antibody Labeling Kits—Table 7.7).
- Zenon Antibody Labeling Kits with Alexa Fluor dye–phycobiliprotein tandem conjugates are also available (Zenon Antibody Labeling Kits—Table 7.7), increasing the possible combinations of detection wavelengths in a multicolor experiment.
Figure 6.4.15 Labeling scheme utilized in the Zenon Antibody Labeling Kits. A) An unlabeled IgG antibody is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled, Fc-specific anti-IgG Fab fragment. B) This labeled Fab fragment binds to the Fc portion of the IgG antibody. C) Excess Fab fragment is then neutralized by the addition of a nonspecific IgG, 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.
For Research Use Only. Not for use in diagnostic procedures.