Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6

Tetramethylrhodamine (TMR) has been an important fluorophore for preparing protein conjugates, especially fluorescent antibody and avidin derivatives used in immunochemistry, although we now strongly recommend using conjugates of our Alexa Fluor 546 and Alexa Fluor 555 dyes (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3) instead of the tetramethylrhodamine conjugates for applications in this spectral range. Under the name TAMRA, the carboxylic acid of TMR has also achieved prominence as a dye for oligonucleotide labeling and automated DNA sequencing applications (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Amine-reactive dyes for nucleic acid sequencing - Table 8.11). Because it can be prepared in high purity, the 5-isomer of TAMRA (C6121) is one of the five dyes in our Reference Dye Sampler Kit (R14782, Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1, Reference Dye Sampler Kit). The detection limit of TMR-labeled amino acids by capillary electrophoresis is reported to be ~600 molecules. The fluorescence quantum yield of TMR conjugates is usually only about one-fourth that of fluorescein conjugates. However, because TMR is readily excited by the intense 546 nm spectral line from mercury-arc lamps used in most fluorescence microscopes and is intrinsically more photostable than fluorescein, TMR conjugates often appear brighter than the corresponding fluorescein conjugates. TMR is also efficiently excited by the 543 nm spectral line of the green He–Ne laser, which is increasingly being used for analytical instrumentation. TMR conjugates are not as well excited by the 568 nm spectral line of the Ar–Kr mixed-gas laser used in many confocal laser-scanning microscopes.

A significant limitation of the TMR dyes TAMRA and TRITC as protein-labeling reagents is that the absorption spectrum of TMR-labeled proteins is frequently complex (Figure 1.71), usually splitting into two absorption peaks at about 520 and 550 nm, so that the actual degree of labeling is difficult to determine. Excitation at wavelengths in the range of the short-wavelength peak fails to yield the expected amount of fluorescence, indicating that it arises from a nonfluorescent dye aggregate. Furthermore, when the TMR-labeled protein conjugate is denatured by guanidine hydrochloride, the long-wavelength absorption increases, the short-wavelength peak mostly disappears and the fluorescence yield almost doubles (Figure 1.71). This change in the absorption spectrum indicates that the extinction coefficient of TMR probably decreases upon conjugation to proteins. The absorption spectra of TMR-labeled nucleotides and of other probes such as our rhodamine phalloidin (R415, Probes for Actin - Section 11.1) do not split into two peaks, indicating a labeling ratio of one dye molecule per biomolecule. The emission spectrum of TMR conjugates does not vary much with the degree of labeling. An improved method for estimating the degree of substitution of TRITC conjugates has been described. Unlike TMR-labeled proteins, protein conjugates of our Alexa Fluor 546 and Alexa Fluor 555 dyes (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3) show normal absorption spectra () and are also more fluorescent than either TMR or Cy3 protein conjugates (Figure 1.22).

Our tetramethylrhodamine isothiocyanate (TRITC) is of the highest quality available from any commercial source. Both our mixed-isomer (T490) and single-isomer (T1480, T1481) TRITC preparations typically have extinction coefficients above 80,000 cm^-1M^-1, whereas some competitive sources of TRITC have extinction coefficients reported to be below 50,000 cm^-1M^-1. TRITC is widely used by other companies to prepare most of their so-called "rhodamine" immunoconjugates; however, they also often employ reactive versions of rhodamine B or Lissamine rhodamine B, which have somewhat different spectra, resulting in some confusion in matching the product name to the correct fluorophore.

Succinimidyl Esters of Carboxytetramethylrhodamine (TAMRA)

Almost all of Molecular Probes' TMR conjugates are prepared using succinimidyl esters of carboxytetramethylrhodamine (TAMRA), rather than TRITC, because bioconjugates from succinimidyl esters are more stable and often more fluorescent. We offer the mixed-isomer (C300) and single-isomer (C6121, C6122) preparations of TAMRA, as well as the corresponding mixed-isomer (C1171) and single-isomer (C2211, C6123) TAMRA succinimidyl esters. The single-isomer preparations of TAMRA are most important for high-resolution techniques such as DNA sequencing and separation of TAMRA-labeled carbohydrates by capillary electrophoresis. 6-TAMRA is one of the traditional fluorophores (5-FAM, 6-JOE, 6-TET, 6-HEX, 6-TAMRA and 6-ROX) used in automated DNA sequencing (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Amine-reactive dyes for nucleic acid sequencing - Table 8.11). Our FluoReporter Tetramethylrhodamine Protein Labeling Kit (F6163, Kits for Labeling Proteins and Nucleic Acids - Section 1.2, FluoReporter(R) Tetramethylrhodamine Protein Labeling Kit) supplies the mixed-isomer 5(6)-TAMRA succinimidyl ester for preparing TMR-labeled proteins.

We have also prepared the mixed-isomer TAMRA-X succinimidyl ester (5(6)-TAMRA-X, SE; T6105), which contains a seven-atom aminohexanoyl spacer ("X") between the reactive group and the fluorophore (). This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated and making it more accessible to secondary detection reagents. Polyclonal anti-tetramethylrhodamine and anti–Texas Red dye antibodies that recognize the tetramethylrhodamine, Rhodamine Red-X, X-rhodamine and Texas Red fluorophores are available (Anti-Dye and Anti-Hapten Antibodies - Section 7.4).

Lissamine Rhodamine B Sulfonyl Chloride

Lissamine rhodamine B sulfonyl chloride (L20, L1908; ) is much less expensive than Texas Red sulfonyl chloride (see below), and the fluorescence emission spectrum of its protein conjugates lies between those of tetramethylrhodamine and Texas Red conjugates (Figure 1.74). Although the absorption spectral shift relative to tetramethylrhodamine is not large, it is sufficient to permit conjugates of Lissamine rhodamine B to be excited by the 568 nm spectral line of the Ar–Kr mixed-gas laser used in many confocal laser-scanning microscopes. Furthermore, the protein conjugates of Lissamine rhodamine B are easier to purify and more chemically stable than are the conjugates of tetramethylrhodamine. Like Texas Red sulfonyl chloride, Lissamine rhodamine B sulfonyl chloride is actually a mixture of isomeric sulfonyl chlorides.

Figure 1.74 Normalized fluorescence emission spectra of goat anti–mouse IgG antibody conjugates of 1) fluorescein, 2) rhodamine 6G, 3) tetramethylrhodamine, 4) Lissamine rhodamine B and 5) Texas Red dyes.

Rhodamine Red-X Succinimidyl Ester

Lissamine rhodamine B sulfonyl chloride is unstable, particularly in aqueous solution, making it somewhat difficult to achieve reproducible conjugations using this dye. Unlike Lissamine rhodamine B sulfonyl chloride, which is a mixture of isomeric sulfonyl chlorides (), our Patented Rhodamine Red-X succinimidyl ester (R6160, ) is isomerically pure. Rhodamine Red-X succinimidyl ester is resistant to hydrolysis at the pH typically used for conjugation and provides a spacer between the fluorophore and the reactive site. Moreover, we have found that protein conjugates of the Rhodamine Red-X dye are frequently brighter than those of Lissamine rhodamine B (Figure 1.76), and are less likely to precipitate during storage. Rhodamine Red-X succinimidyl ester is used in our FluoReporter Rhodamine Red-X Protein Labeling Kit (F6161, FluoReporter(R) Rhodamine Red(R)-X Protein Labeling Kit). See Kits for Labeling Proteins and Nucleic Acids - Section 1.2 for further information on this kit.

Texas Red Sulfonyl Chloride

Texas Red sulfonyl chloride is Molecular Probes' trademarked mixture of isomeric sulfonyl chlorides () of sulforhodamine 101. This reagent is quite unstable in water, especially at the higher pH required for reaction with aliphatic amines. For example, dilute solutions of Texas Red sulfonyl chloride are totally hydrolyzed within 2–3 minutes in pH 8.3 aqueous solution at room temperature. Protein modification by this reagent is best done at low temperature. Once conjugated, however, the sulfonamides that are formed (Figure 1.4) are extremely stable; they even survive complete protein hydrolysis. Because Texas Red sulfonyl chloride rapidly degrades upon exposure to moisture, Molecular Probes offers this reactive dye specially packaged as a set of 10 vials (T1905), each containing approximately 1 mg of Texas Red sulfonyl chloride for small-scale conjugations. We also offer the 10 mg unit size packaged in a single vial (T353) for larger-scale conjugations. Each milligram of Texas Red sulfonyl chloride modifies approximately 8–10 mg of protein. Note that sulfonyl chlorides are unstable in dimethylsulfoxide (DMSO) and should never be used in that solvent. Polyclonal anti-tetramethylrhodamine and anti–Texas Red antibodies that recognize tetramethylrhodamine, Rhodamine Red, X-rhodamine and Texas Red fluorophores are available (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Selected haptenylation reagents and their anti-hapten antibodies - Table 4.2).

Figure 1.4 Reaction of a primary amine with a sulfonyl chloride.

Texas Red-X Succinimidyl Ester

Texas Red sulfonyl chloride's susceptibility to hydrolysis and low solubility in water may complicate its conjugation to some biomolecules. To overcome this difficulty, Molecular Probes has developed and Patented Texas Red-X succinimidyl ester, which contains an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and its reactive group. The single-isomer preparation of Texas Red-X succinimidyl ester (T20175, ) is preferred over the mixed-isomer product (T6134) when the dye is used to prepare conjugates of low molecular weight peptides, oligonucleotides and receptor ligands that are to be purified by high-resolution techniques. Also, because isomers of a reactive dye may differ in their binding geometry, certain applications such as fluorescence resonance energy transfer (FRET) may benefit from the use of single-isomer reactive dyes (Fluorescence Resonance Energy Transfer (FRET) - Note 1.2 ). Thiol-reactive Texas Red derivatives that are based on a similar synthetic approach are described in Thiol-Reactive Probes Excited with Visible Light - Section 2.2. Texas Red-X succinimidyl ester offers significant advantages over Texas Red sulfonyl chloride for the preparation of bioconjugates:

• In the absence of amines, greater than 80% of Texas Red-X succinimidyl ester's reactivity is retained in pH 8.3 solution after one hour at room temperature.
• Much less Texas Red-X succinimidyl ester (usually half or less of the amount of Texas Red sulfonyl chloride) is required to yield the same degree of labeling, making the effective costs of these two reagents about the same.
• Conjugations with Texas Red-X succinimidyl ester are more reproducible.
• Unlike Texas Red sulfonyl chloride, which can form unstable products with tyrosine, histidine, cysteine and other residues in proteins, the Texas Red-X succinimidyl ester reacts almost exclusively with amines.
• Protein conjugates prepared with Texas Red-X succinimidyl ester have a higher fluorescence yield than those with the same labeling ratio prepared with Texas Red sulfonyl chloride (Figure 1.83).
• We have noted a decreased tendency of Texas Red-X protein conjugates to precipitate during the reaction or upon storage.

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

Texas Red-X STP Ester

Molecular Probes has prepared the water-soluble 4-sulfo-2,3,5,6-tetrafluorophenyl (STP) ester of the Texas Red-X dye (T10125). STP Esters, which are prepared by coupling a carboxylic acid and 4-sulfo-2,3,5,6-tetrafluorophenol (S10490, Derivatization Reagents for Carboxylic Acids and Glutamine - Section 3.3, Figure 3.29), react rapidly with amines on proteins (Figure 1.3) under the same conditions as succinimidyl esters but are much more water soluble. STP esters are also available for some of our BODIPY dyes (BODIPY Dye Series - Section 1.4). 

Figure 3.29 4-Sulfo-2,3,5,6-tetrafluorophenol (STP, S10490) can be used to prepare water-soluble activated esters from various carboxylic acids.

Figure 1.3 Reaction of a primary amine with an STP ester

Texas Red C₂-Dichlorotriazine

Texas Red C₂-dichlorotriazine (T30200) is a reactive dye with absorption/emission maxima of ~588/601 nm. Dichlorotriazines readily modify amines in proteins, and they are among the few reactive groups that are reported to react directly with polysaccharides and other alcohols in aqueous solution, provided that the pH is >9 and that other nucleophiles are absent.

Texas Red-X Conjugates and Texas Red-X Labeling Kits

Because of the advantages of Texas Red-X succinimidyl ester, we have converted some of our Texas Red conjugates to the Texas Red-X conjugates. Consequently, we have prepared Texas Red-X conjugates of:

• Antibodies (Secondary Immunoreagents - Section 7.2, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1)
• Streptavidin (S6370, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23)
• Wheat germ agglutinin (W21405, Lectins and Other Carbohydrate-Binding Proteins - Section 7.7)
• dUTP (C7631, Labeling Oligonucleotides and Nucleic Acids - Section 8.2)
• Phalloidin (T7471, Probes for Actin - Section 11.1, Spectral characteristics of our F-actin-selective probes - Table 11.1)
• Polymyxin B (P13237, Viability and Cytotoxicity Assay Reagents - Section 15.2)
• Methotrexate (M23273, Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione - Section 15.6)

Protein conjugates of the Texas Red-X dye are readily prepared using our FluoReporter Texas Red-X Protein Labeling Kit (F6162) and Texas Red-X Protein Labeling Kit (T10244). See Kits for Labeling Proteins and Nucleic Acids - Section 1.2 for further information on these kits. Our Zenon Texas Red-X Antibody Labeling Kits for mouse IgG₁ and rabbit IgG antibodies (Z25045, Z25345; Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3) permit the rapid and quantitative labeling of antibodies from a purified antibody fraction or from a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant with the Texas Red-X dye. Polyclonal anti-tetramethylrhodamine and anti–Texas Red antibodies that recognize tetramethylrhodamine, Rhodamine Red, X-rhodamine and Texas Red fluorophores are available (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Anti-fluorophore antibodies and their conjugates - Table 7.19).

Reactive Texas Red-X dyes and their conjugates are Patented by Molecular Probes. They are offered for research purposes only. We welcome inquiries about Licensing these products for resale or other commercial uses.

The excitation and emission spectra of carboxyrhodamine 6G (CR 6G) fall between those of fluorescein and tetramethylrhodamine (Figure 1.74). With a peak absorption at ~520 nm, conjugates prepared from the mixed-isomer (C6157) or single-isomer (C6127, C6128) preparations of CR 6G succinimidyl esters are an excellent match to the 514 nm spectral line of the argon-ion laser. They also tend to exhibit a higher fluorescence quantum yield than tetramethylrhodamine conjugates, as well as excellent photostability. As with the Rhodamine Green dyes, the carboxyrhodamine 6G dyes are more suitable for preparing nucleotide and oligonucleotide conjugates than for preparing protein conjugates. Oligonucleotide conjugates of the CR 6G dye have spectroscopic and electrophoretic properties that are superior to the JOE dye (C6171MP, Fluorescein, Oregon Green and Rhodamine Green Dyes - Section 1.5) that is often used for DNA sequencing (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Amine-reactive dyes for nucleic acid sequencing - Table 8.11).

One of our reactive BODIPY dyes (BODIPY R6G; D6180, D6186; BODIPY Dye Series - Section 1.4) has spectra similar to carboxyrhodamine 6G but with narrower absorption and emission spectra (Figure 1.41), which may be advantageous for multicolor applications.

Figure 1.41 Normalized fluorescence emission spectra of 1) BODIPY FL, 2) BODIPY R6G, 3) BODIPY TMR, 4) BODIPY 581/591, 5) BODIPY TR, 6) BODIPY 630/650 and 7) BODIPY 650/665 fluorophores in methanol.

Dyes that quench the fluorescence of visible light–excited fluorophores are increasingly important for use in proximity studies (Fluorescence Resonance Energy Transfer (FRET) - Note 1.2 ) and in a wide variety of assays, such as those based on DNA hybridization (Detecting Nucleic Acid Hybridization - Section 8.5). Our QSY 7, QSY 9 and QSY 21 dyes (Molecular Probes' nonfluorescent quenchers and photosensitizers - Table 1.10) are diarylrhodamine derivatives that have several properties that make them superior to the commonly used dabcyl chromophore (Reagents for Analysis of Low Molecular Weight Amines - Section 1.8) when preparing bioconjugates for use in energy transfer–based assays:

• Broad absorption in the visible-light spectrum, with an absorption maximum near 560 nm for both the QSY 7 and QSY 9 dyes and near 660 nm for the QSY 21 dye (Figure 1.70)
• Extinction coefficients that are typically in excess of 90,000 cm^-1M^-1
• Absorption spectra of the conjugates that are insensitive to pH between 4 and 10
• Fluorescence quantum yields typically <0.001 in aqueous solution (In a few isolated cases, we have observed that some QSY dyes can exhibit fluorescence when placed in a rigidifying environment such as glycerol.)
• Efficient quenching of the fluorescence emission of donor dyes by the QSY 7 and QSY 9 dyes, including blue-fluorescent coumarins, any of our green- or orange-fluorescent dyes, and conjugates of the Texas Red and Alexa Fluor 594 dyes
• Quenching of all red-fluorescent dyes by the exceptionally long-wavelength light–absorbing QSY 21 dye (Molecular Probes' amine-reactive dyes - Table 1.1)
• Quenching of most green and red fluorophores that is more effective at far greater distances than is possible with dabcyl quenchers (Molecular Probes' amine-reactive dyes - Table 1.1, Figure 8.51)
• Residual fluorescence of the conjugates, at close spatial separations, that is typically lower than in conjugates that use dabcyl as the quencher
• High chemical stability of the conjugates and very good resistance to photobleaching

The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by fluorescence resonance energy transfer) is defined by the Förster radius (R_o). The magnitude of R_o is dependent on the spectral properties of the donor and acceptor dyes. Förster distances (R_o) calculated for energy transfer from various Alexa Fluor dyes to QSY and dabcyl quenchers are listed in Molecular Probes' amine-reactive dyes - Table 1.1. FRET efficiencies from several donor dyes to the QSY 7 quencher in molecular beacon hybridization probes have also been calculated.

For preparing bioconjugates of the QSY dyes, Molecular Probes offers the amine-reactive QSY 7 (), QSY 9 and QSY 21 succinimidyl esters (Q10193, Q20131, Q20132), the thiol-reactive QSY 7 C₅-maleimide and QSY 9 C₅-maleimide (Q10257, Q30457; Thiol-Reactive Probes Excited with Visible Light - Section 2.2), an aldehyde- and ketone-reactive QSY 9 hydrazide (Q30626) and a QSY 7 aliphatic amine (Q10464, Derivatization Reagents for Carboxylic Acids and Glutamine - Section 3.3) that can be coupled to carboxylic acids and other functional groups. We also have prepared a QSY 7 derivative of α-FMOC lysine (Q21930, Reagents for Peptide Analysis, Sequencing and Synthesis - Section 9.5) for automated synthesis of peptides that contain this important quencher.

These QSY dyes, their conjugates and their use as fluorescence quenchers are Patented by Molecular Probes, Inc. We welcome inquiries about Licensing these products for resale or other commercial uses.

In addition to the QSY 7, QSY 9 and QSY 21 dyes, Molecular Probes has available other quenchers that absorb maximally below 500 nm, including the QSY 35 dye, dabcyl and dabsyl dyes (Molecular Probes' nonfluorescent quenchers and photosensitizers - Table 1.10). These products are described in Reagents for Analysis of Low Molecular Weight Amines - Section 1.8.

Malachite green is a nonfluorescent photosensitizer that absorbs at long wavelengths (~630 nm, ). Its photosensitizing action can be targeted to particular cellular sites by conjugating malachite green isothiocyanate (M689, ) to specific antibodies. Enzymes and other proteins within ~10 Å of the binding site of the malachite green–labeled antibody can then be selectively destroyed upon irradiation with long-wavelength light. Studies by Jay and colleagues have demonstrated that this photoinduced destruction of enzymes in the immediate vicinity of the chromophore is apparently the result of localized production of hydroxyl radicals, which have short lifetimes that limit their diffusion from the site of their generation. Earlier studies had supported a thermal mechanism of action.

In collaboration with Nanoprobes, Inc. (http://www.nanoprobes.com), Molecular Probes offers NANOGOLD and Alexa Fluor FluoroNanogold particles, small metal cluster complexes of gold particles for research applications in light or electron microscopy. The NANOGOLD and Alexa Fluor FluoroNanogold clusters are discrete chemical compounds, not gold colloids. NANOGOLD mono(sulfosuccinimidyl ester) (N20130) permits attachment of these very small (1.4 nm) yet uniformly sized gold particles to biomolecules in the same way that one reacts a succinimidyl ester of a dye (Figure 1.89). This product, which is supplied as a set of five vials of a powder that has been lyophilized from pH 7.5 HEPES buffer, is resuspended with the protein in deionized water at room temperature or below, then any excess NANOGOLD mono(sulfosuccinimidyl ester) is removed by gel filtration and the conjugate is stored frozen (NANOGOLD(R) Mono(sulfosuccinimidyl ester)). 100 nanomoles of NANOGOLD mono(sulfosuccinimidyl ester) is sufficient to label about 100 µg of a protein with a MW of 100,000. Excess reagent should not be stored, and the conjugation mixture must be free of thiols or amine-containing buffers. NANOGOLD and Alexa Fluor FluoroNanogold conjugates of antibodies and streptavidin are described in Secondary Immunoreagents - Section 7.2 and Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, respectively, along with reagents and methods for silver enhancement to amplify electron microscopic detection. In addition to being used for ultrastructural studies, NANOGOLD conjugates are extremely effective excited-state energy transfer quenchers with an enhanced ability to detect single-base mismatches in beacon technology (Figure 8.113). We also supply NANOGOLD monomaleimide (N20345, Thiol-Reactive Probes Excited with Visible Light - Section 2.2, Figure 2.22).

Figure 1.89 Reaction of NANOGOLD mono(sulfosuccinimidyl ester) (N20130) with a primary amine. Image courtesy of Nanoprobes, Inc.

Figure 8.113 Schematic representation of molecular beacons. In the hairpin loop structure, the quencher (black circle) forms a nonfluorescent complex with the fluorophore (green circle). Upon hybridization of the molecular beacon to a complementary sequence, the fluorophore and quencher are separated, restoring the fluorescence.

Figure 2.22 Reaction of NANOGOLD monomaleimide (N20345) with a thiol. Image courtesy of Nanoprobes, Inc.