Fluorescent dyes and nonfluorescent ligands are excellent affinity tags that can be recognized by labeled primary antibodies, providing an alternative to the traditional avidin–biotin system in applications such as in situ hybridization, enzyme-linked immunosorbent assays (ELISAs) and western blot analysis (Biotinylation and Haptenylation Reagents—Section 4.2, Selected haptenylation reagents and their anti-hapten antibodies—Table 4.2). Antibodies to dyes and other ligands provide unique opportunities both for signal enhancement and for correlated fluorescence and electron microscopy studies. Essentially all of the methods that use biotin and avidin reagents (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) are also possible using dyes as haptens, as long as the corresponding anti-dye antibody is also available (Anti-fluorophore and anti-hapten antibodies—Table 7.8).
One advantage of using fluorescent dyes as haptens instead of biotin is that the hapten signal is usually visible, or at least its concentration can be measured by its absorption in solution, preceding the secondary detection step. Unlike biotin, which is an endogenous ligand in mitochondria (), dye-based haptens permit background-free staining of cells and tissues. We provide a large assortment of anti-dye antibodies (Anti-fluorophore and anti-hapten antibodies—Table 7.8), including rabbit polyclonal IgG antibodies to the fluorescein, tetramethylrhodamine, Texas Red, dansyl, Alexa Fluor 488, BODIPY FL, lucifer yellow and Alexa Fluor 405/Cascade Blue fluorophores, as well as a goat IgG anti–fluorescein/Oregon Green dye antibody (A11095). We also provide antibodies against three nonfluorescent haptens: dinitrophenyl (DNP), biotin and nitrotyrosine (Anti-fluorophore and anti-hapten antibodies—Table 7.8).
Antibodies to Fluorescein and Oregon Green Dyes
We have observed complete crossreactivity of our anti-fluorescein antibodies with the Oregon Green 488 and Oregon Green 514 dyes (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5). These antibodies also quench the fluorescence of the structurally similar dye resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1). The high affinity and specificity of anti–fluorescein/Oregon Green dye antibodies (A889, A6413, A6421, A11095) makes fluorescein and Oregon Green dyes ideal haptens for various detection schemes. Researchers have found that fluorescein–anti-fluorescein ELISA techniques display low nonspecific binding and are similar in sensitivity to biotin–streptavidin methods.
Our rabbit polyclonal anti–fluorescein/Oregon Green dye antibody (A889) and goat polyclonal anti–fluorescein/Oregon Green dye antibody (A11095) can be used in combination with any of our Zenon Rabbit IgG and Zenon Goat IgG Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7), respectively, to produce fluorophore-, biotin- or enzyme-labeled antibodies. In addition to these unlabeled anti–fluorescein/Oregon Green dye antibodies, we offer a mouse monoclonal anti–fluorescein/Oregon Green dye antibody and a Fab fragment of rabbit polyclonal anti–fluorescein/Oregon Green dye antibody (Antibody Structure and Classification—Note 7.1). The high-affinity mouse IgG2a monoclonal 4-4-20 anti–fluorescein/Oregon Green dye antibody (A6421) may reduce nonspecific binding in ELISAs and other second-step detection assays. The Fab fragment of our rabbit polyclonal anti–fluorescein/Oregon Green dye antibody (A6413) provides researchers with a probe that more efficiently penetrates cell and tissue preparations. Furthermore, because the Fab fragment no longer contains the Fc portion, nonspecific interactions with Fc receptor–bearing cells are eliminated. As expected, none of our anti–fluorescein/Oregon Green dye antibodies recognize the Alexa Fluor or BODIPY dyes.
We also offer the horseradish peroxidase (HRP) conjugate of the rabbit anti–fluorescein/Oregon Green dye antibody (A21253). HRP conjugates are commonly used in histochemical amplification schemes such as tyramide signal amplification (TSA) technology (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2). TSA technology provides a greater degree of resolution than many conventional enzyme-mediated fluorescent staining methods, and sequential TSA labeling followed by a second round of TSA labeling provides an extremely sensitive assay for detection of low-abundance targets in cells and tissues with high spatial resolution.
Our Alexa Fluor 488 dye–labeled rabbit or goat anti–fluorescein/Oregon Green dye antibodies (A11090, A11096) can be used to enhance the intensity and photostability of the green-fluorescent signal of the fluorescein hapten without changing its fluorescence color. Thus, this conjugate allows researchers to take advantage of the superior photostability of the Alexa Fluor 488 dye, while utilizing existing fluorescein-labeled probes and fluorescein-compatible optics. This strategy has been exploited in our Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes (A11053), which is described in Secondary Immunoreagents—Section 7.2. The Alexa Fluor 594 dye–labeled (A11091) anti–fluorescein/Oregon Green dye antibody can be used to convert the green fluorescence of fluorescein conjugates to photostable red fluorescence, or potentially to amplify the signal from fluorescein conjugates ().
The R-phycoerythrin conjugate of the rabbit IgG anti–fluorescein/Oregon Green dye antibody (A21250) has the unique utility of both shifting the green-fluorescent emission of fluorescein-labeled probes to longer wavelengths and greatly intensifying the long-wavelength signal (Figure 7.4.1). Biotin-XX–labeled rabbit anti–fluorescein/Oregon Green dye antibody (A982) is an excellent reagent for converting a fluorescence-based detection method into an enzyme-amplified light or electron microscopy technique. Biotin-XX anti–fluorescein/Oregon Green can be combined with tyramide signal amplification (TSA) technology (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) in a variety of signal amplification schemes for cell and tissue labeling.
Some of the more important applications for anti–fluorescein/Oregon Green dye antibodies—almost all of which could also be carried out with any of our other anti-dye antibodies and their complementary dyes—include:
- Amplification of the signal from a fluorescein tyramide in TSA protocols (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2)
- Detection of fluorescein-labeled primary antibodies
- Development of fluorescein-labeled cell preparations for electron microscopy
- Investigation of the uptake of a fluorescein dextran in kidney proximal tubules
- Localization of mRNA sequences in a double in situ hybridization experiment in which both fluorescein- and biotin-labeled oligonucleotides were used
- Preparation of an anti-fluorescein affinity matrix, which was used to immobilize a fluoresceinated protein in order to study protein–protein interactions in vitro
- Separation of fluorescein antibody–labeled cell populations by immunoadsorption or magnetic separation techniques
- Assessment of the accessibility of active site–bound fluorescein probes
- Investigation of the internalization pathway of fluorescein transferrin (T2871, Probes for Following Receptor Binding and Phagocytosis—Section 16.1)
Figure 7.4.1 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).
Antibody to the Alexa Fluor 488 Dye
We have prepared a rabbit polyclonal antibody to our green-fluorescent Alexa Fluor 488 dye (A11094). In a manner analogous to the anti–fluorescein/Oregon Green dye antibodies, the anti–Alexa Fluor 488 dye antibody specifically recognizes and efficiently quenches most of the fluorescence of the Alexa Fluor 488 dye. In contrast, the anti–Alexa Fluor 488 dye antibody does not appreciably quench the fluorescence of fluorescein, carboxytetramethylrhodamine or the Alexa Fluor 594 dye. The high affinity of the anti–Alexa Fluor 488 dye antibody makes it potentially useful for various immunochemical applications. This antibody can also be used to further amplify the signals from our Alexa Fluor 488 tyramide–containing TSA Kits (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2).
Antibodies to Tetramethylrhodamine and the Rhodamine Red and Texas Red Dyes
As with the anti–fluorescein/Oregon Green dye antibodies, the rabbit polyclonal antibodies to the tetramethylrhodamine and Texas Red fluorophores (A6397, A6399) are effective reagents for binding these dye-based haptens and quenching their fluorescence. However, these antibodies strongly crossreact with some other rhodamines, including the Rhodamine Red and Lissamine rhodamine B dyes, and therefore cannot be used for simultaneous detection of more than one rhodamine-based dye. These anti-tetramethylrhodamine and anti–Texas Red dye antibodies do not appear to crossreact with fluorescein or the Oregon Green or Alexa Fluor dyes, and our anti–fluorescein/Oregon Green dye antibodies do not crossreact with tetramethylrhodamine or the Rhodamine Red or Texas Red dyes. Anti-tetramethylrhodamine has been used to localize retrogradely transported tetramethylrhodamine dextrans by an immunoperoxidase-based amplification technique.
Antibodies to the Lucifer Yellow Dye and the Cascade Blue and Alexa Fluor 405 Dyes
Lucifer yellow CH (L453) and Cascade Blue hydrazide (C687) are frequently employed as polar tracers for neuronal cell labeling (Polar Tracers—Section 14.3). Our unconjugated (A5750, A5760) and biotinylated (A5751) rabbit polyclonal antibodies to these dyes are useful in standard enzyme-mediated immunohistochemical methods for permanently labeling neuronal tissue. Anti–lucifer yellow dye antibody (A5750) has also been used to follow dye coupling in smooth muscle cells by electron microscopy. The anti–Alexa Fluor 405/Cascade Blue dye antibody (A5760) has been employed in western blot analysis to identify cytoplasmic and luminal domains of the sarcoplasmic reticulum Ca2+-ATPase, which had been photolabeled with Cascade Blue aminoethyl 4-azidobenzamide. The anti–Alexa Fluor 405/Cascade Blue dye antibody also recognizes conjugates of our Alexa Fluor 405 succinimidyl ester (A30000, A30100; Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7), which is a derivative of our Cascade Blue dye with a 4-piperidinecarboxylic acid spacer that separates the fluorophore from its reactive moiety.
Antibody to the BODIPY FL Dye
Our unlabeled rabbit polyclonal antibody to the BODIPY FL fluorophore (A5770) crossreacts with some other BODIPY dyes but does not crossreact appreciably with any other fluorophores. This anti–BODIPY FL dye antibody should therefore not be used for simultaneous detection of more than one dye based on the BODIPY fluorophore. In solution assays, we have found that the anti–BODIPY FL dye antibody effectively quenches most of the fluorescence of the BODIPY FL dye, but quenches the BODIPY TR dye to a lesser degree and does not significantly quench the BODIPY TMR dye. The anti–BODIPY FL dye antibody has been used in a fluorescence quenching assay to determine the accessibility of BODIPY FL dye–labeled cysteine residues in the transmembrane domain of diphtheria toxin.
In contrast to the other anti-fluorophore antibodies, which usually quench the fluorescence of the dye to which they bind, our rabbit polyclonal anti-dansyl antibody (A6398) typically enhances the fluorescence of dansyl amides by greater than 10-fold. Binding of the anti-dansyl antibody also blue shifts the emission spectrum of the fluorophore in water from ~520 nm to ~450 nm. These properties, combined with the unusually high Stokes shift of the dansyl dye (), make this antibody particularly useful for determining the topography of dansyl-labeled probes, including that of dansyl-labeled phospholipids (Fatty Acid Analogs and Phospholipids—Section 13.2) in cell and artificial membranes. The dansyl hapten is preferably incorporated into biomolecules using the succinimidyl ester of dansyl-X (D6104, Biotinylation and Haptenylation Reagents—Section 4.2) because its aminohexanoyl spacer ("X") reduces the interaction of the fluorophore with the biomolecule to which it is conjugated and makes it more accessible to anti-dansyl antibodies.
Except for the anti-dansyl antibody, which enhances the fluorescence of the dansyl fluorophore, all of our anti-fluorophore antibodies strongly quench the fluorescence of their complementary dyes in free solution. For example, our anti–fluorescein/Oregon Green dye antibodies typically effect up to 95% quenching of the fluorescence of both fluorescein and the Oregon Green 488 dye. The anti–fluorescein/Oregon Green dye antibody also quenches some other fluorescein derivatives, such as carboxyfluorescein, Calcium Green-1 Ca2+ indicator and BCECF pH indicator, making this antibody useful for reducing background fluorescence caused by leakage of these dyes from the cell. However, quenching of our fluorescein-based Ca2+ indicators by our anti–fluorescein/Oregon Green dye IgG antibody is apparently dependent on whether or not Ca2+ is bound; Calcium Green-1 indicator is quenched by 89% in the presence of 5 µM Ca2+, whereas it is quenched by only 46% in the presence of 10 mM EGTA. Maximal quenching efficiencies for fluorescein analogs (all at 5 nM dye using the rabbit IgG anti–fluorescein/Oregon Green dye antibody, A889) are as follows (values may vary somewhat from batch to batch) and may be different using the goat IgG anti–fluorescein/Oregon Green dye antibody, A11095, the mouse monoclonal anti–fluorescein/Oregon Green dye, A6421 or the rabbit IgG Fab fragment of anti–fluorescein/Oregon Green dye, A6413):
- Oregon Green 488 dye, 95%
- Oregon Green 514 dye, 92%
- Carboxyfluorescein, 93%
- Calcium Green-1 indicator (in the presence of 5 µM Ca2+), 89%
- Calcium Green-1 indicator (in the presence of 10 mM EGTA), 46%
- BCECF indicator, 43%
- Fluo-3 indicator (in the presence of 5 µM Ca2+), 32%
- Rhodamine Green dye, 9%
- Calcein, <5%
- Tetramethylrhodamine, <5%
Our preparations of the anti-tetramethylrhodamine and anti–Texas Red dye antibodies are somewhat less effective as fluorescence quenchers of their complementary fluorophores, with maximal quenching efficiencies of ~75% and ~60%, respectively. Our rabbit IgG anti–Alexa Fluor 488 dye antibody quenches the fluorescence of the free Alexa Fluor 488 dye by >90%. Our antibody to the BODIPY FL fluorophore typically quenches the dye's fluorescence by ~90%. It also quenches BODIPY TR dye fluorescence by ~45%, but does not significantly quench BODIPY TMR dye fluorescence. Antibodies to the lucifer yellow and Alexa Fluor 405/Cascade Blue fluorophores quench the fluorescence of their complementary dyes by ~85% and ~80%, respectively. In addition, anti-DNP antibodies have been reported to significantly quench the fluorescence of aminonitrobenzoxadiazoles (NBD amines).
We use a sensitive fluorescence quenching–based assay to ensure that the concentration of specific anti-dye antibody in its purified IgG fractions is provided at a consistently high titer. As supplied, 20 µL of the antibody solution is certified to produce ≥50% of the maximal fluorescence quenching (or enhancement, in the case of anti-dansyl antibody) of 1 mL of a 50 nM solution of the corresponding dye, assayed in 100 mM sodium phosphate, pH 8.0. All maximal quenching values are determined using the free fluorophore; the maximal quenching of a fluorophore covalently bound to a protein is often significantly less due to steric hindrance.
Applications for Fluorescence Quenching by Anti-Fluorophore Antibodies
Fluorescence quenching of dye haptens by anti-dye antibodies provides a useful measure of topography in cells, proteins and membranes. For example, researchers have used anti-fluorescein quenching assays to determine the accessibility of a fluorescein-labeled ATP-binding site in both Na+/K+-ATPase and Ca2+/Mg2+-ATPase. Similarly, the anti–BODIPY FL dye antibody has been employed to identify shallow- and deep-membrane–penetrating forms of diphtheria toxin T domain. In addition, anti-fluorophore antibodies have been used as cell-impermeant probes for determining whether fluorescent dye–conjugated ligands, proteins, bacteria or other biomolecules have been internalized by endocytic or pinocytic processes (Probes for Following Receptor Binding and Phagocytosis—Section 16.1). Anti-fluorophore antibodies also permit background-free observation of fusion events in an assay designed to monitor the fusion of membrane vesicles in vitro. As noted above, however, these antibodies may quench dye-labeled proteins less effectively than they quench free dyes.
Because of its high affinity for the dinitrophenyl (DNP) hapten, our anti-DNP polyclonal rabbit antibodies (Anti-fluorophore and anti-hapten antibodies—Table 7.8) are excellent reagents for probing DNP-labeled molecules, including nucleic acid probes prepared using DNP-labeled nucleotides. Unlike assays that use biotin as the hapten, it is usually easy to determine the degree of substitution of the DNP hapten in bioconjugates from the dye's visible absorption near 350 nm (EC
In addition to the unlabeled anti-DNP antibody (A6430), we offer anti-DNP antibody conjugates of biotin-XX (A6435), fluorescein (A6423) and Alexa Fluor 488 dye (A11097). Our anti-DNP antibody is prepared against DNP–keyhole limpet hemocyanin (DNP-KLH) and thus the antibody and its conjugates do not crossreact with BSA, a common blocking reagent in hybridization applications.
For use in conjunction with our anti-DNP antibody, we offer the DNP-X succinimidyl ester (D2248, Biotinylation and Haptenylation Reagents—Section 4.2) for labeling proteins and amine-modified DNA. The literature also describes methods for incorporating DNP into DNA using DNP-labeled primers. In addition to recognizing DNP, our anti-DNP antibody crossreacts with trinitrobenzenesulfonic acid–modified proteins, making this antibody useful both for localizing and for isolating cell-surface molecules labeled with either DNP or TNP. Furthermore, anti-DNP antibodies have been reported to quench aminonitrobenzoxadiazoles (NBD amines), indicating that NBD-X succinimidyl ester (S1167, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7) will also be a useful haptenylating reagent for use with this antibody.
Our antibody to nitrotyrosine (A21285) is raised in rabbits that have been immunized with nitrated KLH. Nitrotyrosine-modified proteins are the principal reaction products of nitric oxide (through the formation of peroxynitrite) in cells (Probes for Nitric Oxide Research—Section 18.3). Because tyrosine residues are also conveniently converted to nitrotyrosine by reaction at near-neutral pH with tetranitromethane, the nitrotyrosine hapten can be readily created in almost any peptide or protein that contains a tyrosine residue. A further advantage is that nitrotyrosine has pH-dependent visible absorbance (absorption maxima ~360 nm and 428 nm ) that can be utilized to detect formation of the hapten in soluble biopolymers.
Our anti-nitrotyrosine antibody is useful for detecting nitrotyrosine-containing peptides and proteins both in immunohistochemical () and western blot applications (Figure 7.4.2). It can be used to identify nitrated proteins and to determine the level of protein nitrosylation in tissues. This rabbit IgG antibody can be labeled with fluorophores, biotin or enzymes using any of our Zenon Rabbit IgG Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7).
Figure 7.4.2 Specificity of our rabbit anti-nitrotyrosine antibody (A21285) to nitrated proteins. Equal amounts of avidin (A887, lane 1) and CaptAvidin biotin-binding protein (C21385, lane 2) were run on an SDS-polyacrylamide gel (4–20%) and blotted onto a PVDF membrane for western blot analysis. CaptAvidin biotin-binding protein, a derivative of avidin, has nitrated tyrosine residues in the biotin-binding site. Nitrated proteins were identified with the anti-nitrotyrosine antibody, in combination with an alkaline phosphatase conjugate of goat anti–rabbit IgG antibody (G21079) and the red-fluorescent substrate, DDAO phosphate (D6487).
The high affinity of avidin for biotin was first exploited in histochemical applications in the early 1970s. The use of avidin–biotin techniques has since become standard for diverse detection schemes, although limitations of this method have also been recognized. As an alternative to avidin-based reagents, we offer a high-affinity mouse monoclonal antibody to biotin (03-3700). This anti-biotin antibody can be used to detect biotinylated molecules in immunohistochemistry, in situ hybridization, ELISAs and flow cytometry applications.
It has been shown that certain monoclonal antibodies to biotin have biotin-binding motifs that are similar to those seen for avidin and streptavidin. Anti-biotin antibody has been shown to selectively stain endogenous biotin-dependent carboxylase proteins used in fatty acid synthesis of the mitochondria. Nonspecific staining of mitochondrial proteins by labeled avidins and by anti-biotin antibodies can be a complicating factor in using avidin–biotin techniques (). This nonspecific binding can usually be blocked by pretreatment of the sample with the reagents in our Endogenous Biotin-Blocking Kit (E21390, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6).