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Molecular Probes The Handbook

BODIPY Dye Series—Section 1.4

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Overview of Our BODIPY Fluorophores

Our Patented BODIPY fluorophores have spectral characteristics that are often superior to those of fluorescein, tetramethylrhodamine, Texas Red and longer-wavelength dyes and may be substituted for these dyes in some applications. With derivatives that span the visible spectrum (Figure 1.41), BODIPY dyes are proving to be extremely versatile. We use them to generate fluorescent conjugates of proteins, nucleotides, oligonucleotides and dextrans, as well as to prepare fluorescent enzyme substrates, fatty acids, phospholipids, lipopolysaccharides, receptor ligands and polystyrene microspheres. BODIPY dyes are unusual in that they are relatively nonpolar and the chromophore is electrically neutral (Figure 1.42). These properties sometimes enhance the affinity of their ligand conjugates for receptors, as long as the overall conjugate is not too lipophilic. BODIPY dye conjugates of low molecular weight molecules also tend to be more permeant to live cells than are conjugates of charged fluorophores (Membrane-Permeant Reactive Tracers - Section 14.2). In addition, oligonucleotide conjugates of several of our BODIPY dyes have been reported to be useful for DNA sequencing ref (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Specialty nucleic acid reagents for molecular biology - Table 8.1), in part because the dye exhibits minimal effect on the mobility of the fragment during electrophoresis. And with their high peak intensity, reactive BODIPY dyes are among the most detectable amine-derivatization reagents available for HPLC and capillary electrophoresis.ref The BODIPY dyes are more useful than most other long-wavelength dyes, including fluoresceins and carbocyanines, for assays that measure fluorescence polarization ref (Fluorescence Polarization (FP) - Note 1.5). Furthermore, BODIPY dyes have exceptionally large cross-sections for excitation by multiphoton excitation sources.ref




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.




Figure 1.42 The structure and numbering of the BODIPY fluorophore, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene.


Amine-reactive BODIPY dyes (Amine-reactive BODIPY dyes - Table 1.7) are discussed below; thiol-reactive BODIPY dyes are included in Thiol-Reactive Probes Excited with Visible Light - Section 2.2. Other reactive BODIPY dyes useful for derivatizing aldehydes, ketones and carboxylic acids are described in Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2 and Derivatization Reagents for Carboxylic Acids and Glutamine - Section 3.3. Applications of some thiol-reactive BODIPY dyes for cell tracing are discussed in Membrane-Permeant Reactive Tracers - Section 14.2.

The core structure of the BODIPY fluorophore is shown in Figure 1.42. Solutions of the alkyl-substituted derivatives have a green, fluorescein-like fluorescence. However, when substituents that yield additional conjugation are added to the parent molecule, both the absorption and emission spectra of the resulting derivatives can shift to significantly longer wavelengths, with emission maxima of greater than 750 nm now possible with some BODIPY derivatives. Our goal has been to develop BODIPY dyes that are optimal for the major excitation sources and that match the common optical filter sets (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11). Accordingly, our best BODIPY substitutes for the fluorescein, rhodamine 6G, tetramethylrhodamine and Texas Red fluorophores are BODIPY FL, BODIPY R6G, BODIPY TMR and BODIPY TR, respectively (Figure 1.43). Because there are so many BODIPY dyes, we have had to develop a systematic strategy for naming them. Except for BODIPY FL, BODIPY R6G, BODIPY TMR and BODIPY TR, we now identify these dyes with the registered trademark BODIPY followed by the approximate absorption/emission maxima in nm (determined in methanol); for example, the BODIPY 581/591 dye.

The BODIPY fluorophores, reactive dyes and conjugates are covered by several Patents issued to Molecular Probes. These products are offered for research purposes only. Molecular Probes welcomes inquiries about Licensing these products for resale or other commercial uses. Custom conjugations of the various BODIPY fluorophores are also available from Molecular Probes; please contact our Custom and Bulk Sales Department. Custom-conjugated oligonucleotides are available through our licensees (Licensing).

BODIPY FL Dye: A Substitute for Fluorescein

With the most fluorescein-like spectra of the BODIPY dyes, the green-fluorescent BODIPY FL fluorophore (spectra) (excitation/emission maxima ~503/512 nm) has several characteristics ref that make it potentially superior to fluorescein in some applications. These include:

  • High extinction coefficient (ε >80,000 cm-1M-1)
  • High fluorescence quantum yield (often approaching 1.0, even in water)
  • Spectra that are relatively insensitive to solvent polarity and pH ref
  • Narrow emission bandwidth (Figure 1.43), resulting in a higher peak intensity than that of fluorescein
  • Unique red shift in fluorescence emission at high dye concentrations — a property that can be used to detect regions of high probe density (photo)
  • Relatively long excited-state lifetime (typically 4 nanoseconds or longer), making the BODIPY FL dye useful for fluorescence polarization–based assays (Fluorescence Polarization (FP) - Note 1.5)
  • Little or no spectral overlap with longer-wavelength dyes such as tetramethylrhodamine and Texas Red dye (Figure 1.43), making BODIPY FL one of the preferred green-fluorescent dyes for multicolor applications ref (photo)
  • Greater photostability than fluorescein in some environments ref (Figure 1.46)
  • A large two-photon cross-section for multiphoton excitation ref (photo)
  • Lack of ionic charge



Figure 1.43 Normalized fluorescence emission spectra of goat anti–mouse IgG antibody conjugates of fluorescein (FL), tetramethylrhodamine (TMR) and the Texas Red (TR) dyes, shown by dashed lines (---), as compared with goat anti–mouse IgG antibody conjugates of BODIPY FL, BODIPY TMR and BODIPY TR dyes, respectively, shown by solid lines (—).



Figure 1.46 Comparison of photostability of green-fluorescent antibody conjugates. The following fluorescent goat anti–mouse IgG antibody conjugates were used to detect mouse anti–human IgG antibody labeling of human anti-nuclear antibodies in HEp-2 cells on prefixed test slides (INOVA Diagnostics Corp.): Oregon Green 514 (O6383, filled square), Alexa Fluor 488 (A11001, open circle), BODIPY FL (B2752, open triangle), Oregon Green 488 (O6380, open square) or fluorescein (F2761, filled circle). Samples were continuously illuminated and viewed on a fluorescence microscope using a fluorescein longpass filter set. Images were acquired every 5 seconds. For each conjugate, three data sets, representing different fields of view, were averaged and then normalized to the same initial fluorescence intensity value to facilitate comparison.

Longer-Wavelength BODIPY Dyes

We have found that it is possible to synthesize BODIPY fluorophores with altered spectral properties by simply changing the substituents on the parent molecule. This discovery has led to creation of a series of longer-wavelength BODIPY dyes with fluorescence spectra that span the visible spectrum (Figure 1.41). The BODIPY R6G (excitation/emission maxima ~528/547 nm), BODIPY TMR (excitation/emission maxima ~543/569 nm) and BODIPY TR (excitation/emission maxima ~592/618 nm) fluorophores are spectrally similar to the rhodamine 6G (R634, Probes for Mitochondria - Section 12.2), tetramethylrhodamine and Texas Red fluorophores, respectively, and are thus compatible with standard optical filter sets designed for these important dyes (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11). The red fluorescence of the BODIPY 581/591 fluorophore shifts to green fluorescence upon peroxidation, a feature that has been exploited for ratiometric measurements of lipid oxidation in live cells ref (Generating and Detecting Reactive Oxygen Species - Section 18.2). The BODIPY 630/650-X and BODIPY 650/665-X fluorophores are the longest-wavelength amine-reactive BODIPY fluorophores currently available. The spectral properties of these longer-wavelength BODIPY derivatives retain most of the advantages of the BODIPY FL fluorophore, including narrow bandwidths, high extinction coefficients, good fluorescence quantum yields and relatively long excited-state lifetimes (>3 nanoseconds for the BODIPY 630/650 dye ref). Like the BODIPY FL fluorophore, however, most of these dyes have a small Stokes shift, which may require that they be excited or detected at suboptimal wavelengths. Nevertheless, even when suboptimal excitation is required, the BODIPY dyes are among the most intensely fluorescent dyes available. The spectral characteristics of 13 different red-fluorescent fluorophores, including the Alexa Fluor 647 (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3) and BODIPY 630/660 dyes, have been evaluated in different surrounding media to assess the influence of polarity, viscosity and detergent concentration and to facilitate probe choice in fluorescence-based assays.ref

Amine-Reactive BODIPY Dyes

BODIPY Dye Succinimidyl Esters

Molecular Probes offers an extensive selection of amine-reactive BODIPY dyes (Amine-reactive BODIPY dyes - Table 1.7), including succinimidyl esters of several BODIPY propionic acids and of one BODIPY pentanoic acid. In addition, we have prepared the reactive succinimidyl esters of:


These reactive dyes contain an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and the succinimidyl ester group. 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 such as anti-dye antibodies.ref BODIPY TMR-X SE has been conjugated to a series of peptide ligands for use in a high-throughput fluorescence polarization assay of ligand binding to G protein–coupled receptors.ref Several BODIPY succinimidyl esters have been conjugated to aminoacyl tRNAs for metabolic incorporation into proteins through in vitro translation.ref For amplifying the BODIPY FL dye signal or converting it into an electron-dense signal, we offer an unlabeled anti–BODIPY FL rabbit polyclonal antibody (A5770, Anti-Dye and Anti-Hapten Antibodies - Section 7.4). This antibody crossreacts with some other BODIPY dyes, but not with other fluorophores, and therefore should not be used for simultaneous detection of more than one dye based on the BODIPY fluorophore.

The BODIPY propionic acid succinimidyl esters (D2184, D2187, D2191, D2219, D2222, D2225, D2228, D6180) and BODIPY FL pentanoic acid succinimidyl ester (D6184) are particularly useful for preparing conjugates of peptides, nucleotides, oligonucleotides,ref drugs, toxins, sphingolipids and other low molecular weight ligands that contain aliphatic amines and for derivatizing amines in various high-resolution separation technologies (Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores - Section 1.7). Our BODIPY 630/650-X and BODIPY 650/665-X succinimidyl esters (D10000, D10001) are quite fluorescent when conjugated to nucleotides ref and oligonucleotides, and can be excited by near-infrared excitation sources. In addition to their use in preparing bioconjugates, at least two of our BODIPY dye succinimidyl esters — BODIPY FL-X SE and BODIPY TR-X SE — are very useful for quantitative and extremely sensitive protein staining on blots and arrays, and we have therefore utilized these amine-reactive BODIPY derivatives in our DyeChrome Western Blot Stain Kits (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4, photo).

Water-Soluble BODIPY FL Succinimidyl Esters and STP Esters

The moderate lipophilicity of the BODIPY propionic acid succinimidyl esters discussed above requires their dissolution in an organic solvent before use in conjugations. Although these reactive dyes are very useful for preparing conjugates of amines in organic solvents, they are less suitable for use with proteins than our water-soluble amine-reactive BODIPY dyes. We usually use the succinimidyl ester of BODIPY FL cysteic acid (BODIPY FL, CASE; D6141), which contains a sulfonated spacer that appears to decrease the interaction between the fluorophore and the protein (structure) and thus reduce the quenching in our protein conjugates. Both this cysteic acid derivative and the sulfosuccinimidyl ester of BODIPY FL propionic acid (BODIPY FL, SSE; D6140) are quite soluble in water and potentially useful for preparing conjugates of most proteins, amine-modified oligonucleotides and other biomolecules. Sulfosuccinimidyl esters of biotin (B6352, B6353; Biotinylation and Haptenylation Reagents - Section 4.2) are sometimes employed as cell-impermeant probes for selectively labeling the outer membrane of cells in topological studies;ref these sulfonated BODIPY FL succinimidyl esters and the STP esters described below may be similarly useful.

Molecular Probes has introduced water-soluble STP Esters ref of some of our most popular BODIPY dyes:

  • BODIPY FL, STP ester (B10006)
  • BODIPY TMR, STP ester (B10002)
  • BODIPY TR-X, STP ester (B10003)
  • BODIPY 650/665-X, STP ester (B10005)

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 1.3), are more readily purified than sulfosuccinimidyl esters but equally amine reactive. They are more suitable than the corresponding BODIPY dye succinimidyl esters for amine conjugation in aqueous solution.




Figure 1.3 Reaction of a primary amine with an STP ester.


BODIPY Carboxylic Acids

Two green-fluorescent BODIPY carboxylic acids (D2183, D3834) are available. These carboxylic acid derivatives can be converted to fluorescent esters,ref acid halides or amides using standard chemical techniques.

BODIPY Dye Conjugates

The versatility of the BODIPY fluorophore is demonstrated by its incorporation into literally hundreds of products listed in this Handbook, including many of our FluoSpheres and TransFluoSpheres microspheres (Microspheres - Section 6.5), enzyme substrates (Enzyme Substrates - Chapter 10) and several of our imaging and flow cytometry standards (Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1, Flow Cytometry Reference Standards - Section 23.2).

Peptides and Proteins

As is common with many fluorescent dyes, conjugation of BODIPY dyes to proteins is sometimes accompanied by significant fluorescence quenching. Because of this potential problem, we do not recommend using the simple BODIPY propionic acid succinimidyl esters discussed above for preparing most protein conjugates, although peptides labeled with a single BODIPY dye can be quite fluorescent and are quite useful for fluorescence polarization–based assays ref (Labeling Small Peptides with Amine Reactive Dyes in Organic Solvents - Note 9.2). Molecular Probes prepares conjugates of its BODIPY dyes with an exceptionally wide variety of peptides, proteins and polysaccharides, including:


In addition, Molecular Probes prepares conjugates of proteins (and of starch) that are so heavily labeled that they are almost nonfluorescent (Figure 10.56). Use of the EnzChek Kits and DQ reagents that incorporate these bioconjugates as fluorogenic enzyme substrates is described later in this section and in Detecting Peptidases and Proteases - Section 10.4.




Figure 10.56 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hydrolysis of the heavily labeled and almost totally quenched substrates provided in our EnzChek Protease Assay Kits (E6638, E6639, E33757), EnzChek Ultra Amylase Assay Kit (E33651), EnzChek Gelatinase/Collagenase Assay Kit (E12055), EnzChek Elastase Kit (E12056), EnzChek Lysozyme Assay Kit (E22013) — as well as the stand-alone quenched substrates DQ BSA (D12050, D12051), DQ collagen (D12052, D12060), DQ ovalbumin (D12053) and DQ gelatin (D12054) — relieves the intramolecular self-quenching, yielding brightly fluorescent reaction products.


BODIPY Dye Conjugates of Nucleotides and Oligonucleotides

With the exception of guanosine nucleotides (see below), fluorescence quenching is usually not a problem if the BODIPY derivative is conjugated to nucleotides, oligonucleotides, peptides or low molecular weight amines in which the stoichiometry of modification is 1:1. Oligonucleotide conjugates of the BODIPY dyes are among the brightest derivatives available for DNA sequencing, nucleic acid hybridization ref and other applications. BODIPY FL dye–labeled oligonucleotide primers also have lower photodestruction rates than fluorescein-labeled primers, improving the detectability of labeled DNA in sequencing gels.ref Oligonucleotide conjugates of several of our BODIPY dyes have been shown to be useful for DNA sequencing ref (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Amine-reactive dyes for nucleic acid sequencing - Table 8.11), in part because the dye exhibits minimal effect on the mobility of the fragment during electrophoresis. Molecular Probes also offers an assortment of BODIPY dye–labeled ChromaTide nucleotides for enzyme-mediated incorporation into nucleic acids (Labeling Oligonucleotides and Nucleic Acids - Section 8.2; Characteristics of ChromaTide UTP nucleotides - Table 8.6, Characteristics of ChromaTide dUTP, ChromaTide OBEA-dCTP, aha-dUTP and aha-dCTP labeled nucleotides - Table 8.7) or for use as structural probes of nucleotide-binding proteins (Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins - Section 17.3).

BODIPY Dye Conjugates of Lipids and Receptor Ligands

BODIPY dye conjugates of other molecules — lipids, toxins, steroids, drug analogs, receptor probes, enzyme substrates and the like — often have quantum yields approaching unity, especially in organic solvents. The lack of ionic charge makes several of these derivatives useful for staining receptors in live cells.

Our BODIPY derivatives of low molecular weight ligands include analogs of:


In addition to the BODIPY dye conjugates of receptor ligands in the list above, we have utilized BODIPY dyes for synthesis of several LysoTracker (photo) and LysoSensor dyes, as well as BODIPY FL histamine (B22461, photo), that are extremely useful probes for labeling acidic organelles in live cells. These products are discussed in Probes for Lysosomes, Peroxisomes and Yeast Vacuoles - Section 12.3.

BODIPY Dye Conjugates as Enzyme Substrates and for High-Throughput Screening Applications

EnzChek Kits and DQ Reagents as Fluorogenic Enzyme Substrates

We have found BODIPY dye conjugates to be very useful reagents for numerous bioanalytical screening applications. In particular, we have utilized the tendency of BODIPY dyes to quench their fluorescence on conjugation to certain biopolymers to our advantage (Figure 10.56) in the following enzyme-assay kits and reagents:




Figure 16.5 Immune complex of DQ BSA conjugate (D12050, D12051) with an anti–bovine serum albumin (BSA) antibody (A11133) for the fluorescent detection of the Fc receptor–mediated phagocytosis pathway. The DQ BSA is a derivative of BSA that is labeled to such a high degree with either the green-fluorescent BODIPY FL or red-fluorescent BODIPY TR-X dye that the fluorescence is self-quenched. Upon binding to an Fc receptor, the nonfluorescent immune complex is internalized and the protein is subsequently hydrolyzed to fluorescent peptides within the phagovacuole.

Conjugation of either the BODIPY FL dye (excitation/emission maxima ~500/506 nm) or BODIPY TR dye (excitation/emission maxima ~589/617 nm) to a biopolymer at high degrees of substitution (DOS) results in almost total quenching of the conjugate's fluorescence; they typically exhibit <3% of the fluorescence of the corresponding free dyes. Enzyme-catalyzed hydrolysis relieves this quenching, yielding brightly fluorescent BODIPY FL dye– or BODIPY TR-X dye–labeled peptides (Figure 10.56), or, in the case of the BODIPY FL amylase substrate in the EnzChek Amylase Assay Kit, BODIPY FL dye–labeled carbohydrates. The increase in fluorescence, which can be measured with a spectrofluorometer, minifluorometer or fluorescence microplate reader, is proportional to enzymatic activity. The DQ ovalbumin and DQ BSA substrates are particularly suitable for the study of receptor labeling and antigen processing. Ovalbumin is efficiently processed through mannose receptor–mediated endocytosis by antigen-presenting cells and is widely used for studying antigen processing. The BSA conjugates can be targeted to Fc receptors by complexing the DQ BSA reagents with our anti-BSA antibody (A11133, Primary Antibodies for Diverse Applications - Section 7.5). Upon endocytosis and proteolysis, highly fluorescent peptides are released within intracellular vacuoles. DQ ovalbumin appears to be an excellent indicator of macrophage-mediated antigen processing in flow cytometry or microscopy assays.

EnzChek Polarization Assay Kit for Proteases

When a fluorescent molecule tethered to a protein is excited by polarized fluorescent light, the polarization of fluorescence emission is dependent on the rate of molecular tumbling. Upon proteolytic cleavage of the fluorescently labeled protein, the smaller peptides that result tumble faster and the emitted light is depolarized relative to the light measured from the intact conjugate (Fluorescence Polarization (FP) - Note 1.5). The EnzChek Polarization Assay Kit for Proteases (E6658, Detecting Peptidases and Proteases - Section 10.4, EnzChek(R) Polarization Assay Kit for Proteases) contains green-fluorescent BODIPY FL casein with an optimal degree of labeling for fluorescence polarization–based protease assays. Fluorescence polarization technology is more sensitive than many other nonradioactive assays for proteases and allows measurements to be taken in real time, permitting the collection of kinetics data. Our BODIPY FL dye has an adequate fluorescence lifetime and pH-insensitive fluorescence — two prerequisites for successful measurement of protease activity by fluorescence polarization.

Lipophilic BODIPY Substrates for Phospholipases and Other Enzymes

The low polarity of the BODIPY fluorophore makes probes containing these dyes excellent analogs of biological lipids (Probes for Lipids and Membranes - Chapter 13). Consequently, these probes are well tolerated by enzymes that metabolize lipids, including phospholipases and enzymes that act on sphingolipids (Probes for Lipid Metabolism and Signaling - Section 17.4, Fluorescence-based phospholipase assays - Table 17.2). In most cases, lack of a spectral shift in the metabolic product's fluorescence requires use of an easy extraction and chromatographic separation step to detect product formation, with quantitation possible by photography or with a fluorescence- or absorption-based scanner. Useful lipophilic substrates based on our BODIPY dyes include:




Figure 17.24 Mechanism of phospholipase activity–linked fluorescence enhancement responses of bis-BODIPY FL C11-PC (B7701) and PED6 (D23739). Note that enzymatic cleavage of bis-BODIPY FL C11-PC yields two fluorescent products, whereas cleavage of PED6 yields only one.


BODIPY Dye–Based Substrates for Chloramphenicol Acetyltransferase

Chloramphenicol acetyltransferase (CAT), an enzyme that is encoded by an important reporter gene, can acetylate chloramphenicol derivatives that incorporate the BODIPY fluorophore (Figure 10.71). The acetylated products are readily separated from the substrate by thin-layer chromatography (photo) and quantitated by photography, fluorometry or with a plate scanner. Our original FAST CAT Chloramphenicol Acetyltransferase Assay Kit ref (F2900) and improved FAST CAT (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6616) utilize a green-fluorescent BODIPY FL substrate and the FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6617) utilizes a yellow-fluorescent BODIPY TMR 1-deoxychloramphenicol substrate. These products are described in detail in Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes - Section 10.6 and in the associated product literature (FAST CAT(R) Chloramphenicol Acetyltransferase Assay Kit, FAST CAT(R) (deoxy) Chloramphenicol Acetyltransferase Assay Kits).




Figure 10.71 The green-fluorescent BODIPY FL 1-deoxychloramphenicol substrate in our FAST CAT Green (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6616). CAT-mediated acetylation of this substrate and of the BODIPY TMR 1-deoxychloramphenicol in our FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6617) results in single fluorescent products because these substrates contain only one hydroxyl group that can be acetylated. In contrast, the BODIPY FL chloramphenicol substrate in our original FAST CAT Kit (F2900) contains a second hydroxyl group at the 1-position (indicated by the labeled arrow). This hydroxyl group undergoes a nonenzymatic transacetylation step, restoring the original hydroxyl for a second acetylation. CAT-mediated acetylation of this chloramphenicol substrate produces three fluorescent products, thus complicating the analysis.

BODIPY Dye–Labeled Nucleotides as Enzyme Substrates and for High-Throughput Screening Applications

Molecular Probes offers BODIPY dye–labeled ChromaTide nucleotides, including both UTP and dUTP derivatives (Labeling Oligonucleotides and Nucleic Acids - Section 8.2; Characteristics of ChromaTide UTP nucleotides - Table 8.6, Characteristics of ChromaTide dUTP, ChromaTide OBEA-dCTP, aha-dUTP and aha-dCTP labeled nucleotides - Table 8.7). These nucleotide analogs are readily incorporated into DNA or RNA by a variety of enzymes. Among these derivatives is the BODIPY FL-14-dUTP conjugate (C7614, Labeling Oligonucleotides and Nucleic Acids - Section 8.2), which has been used as a terminal deoxynucleotidyl transferase (TdT) substrate in the detection of apoptotic cells (TUNEL assay, Assays for Apoptosis - Section 15.5; photo).

In addition to the ChromaTide nucleotides labeled with BODIPY dyes, we have prepared BODIPY FL conjugates of ATP, AMPPNP, GTP and GMPPNP that are labeled through the ribose moieties (A12410, B22355, G12411, B22356; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins - Section 17.3). In this case, BODIPY FL GTP and BODIPY FL GMPPNP both show significant fluorescence quenching (Figure 17.16) that is relieved by binding to GTP-binding proteins (G-proteins). Longer-wavelength BODIPY R6G and BODIPY TR conjugates of ATP and GTP are also available (A22352, G22350, G22351; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins - Section 17.3).





Figure 17.16 Fluorescence emission spectra of (1) free BODIPY FL dye in phosphate-buffered saline, pH 7.2; (2) BODIPY FL ATP (A12410); and (3) BODIPY FL GTP (G12411). Samples were prepared with equal absorbance at the excitation wavelength (488 nm). The areas under the curves are therefore proportional to the relative fluorescence quantum yields, clearly showing the quenching effect caused by interaction of the BODIPY FL fluorophore with the guanine base of GTP.


For protein-binding studies that require nonhydrolyzable nucleotides, we offer the BODIPY FL fluorophore linked through the γ-thiol of ATP-γ-S (A22184, structure) and the BODIPY FL, BODIPY 515/530 and BODIPY TR fluorophores linked through the γ-thiol of GTP-γ-S ref (G22183, G35779, G35780; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins - Section 17.3). Like BODIPY FL GMPPNP, the fluorescence of the BODIPY GTP-γ-S thioesters is quenched ~90% relative to that of the free dye but is recovered upon protein binding to at least some G-proteins.ref The green-fluorescent BODIPY FL GTP-γ-S has been used to detect GTP-binding proteins separated by capillary electrophoresis.ref BODIPY 515/530 GTP-γ-S thioester also exhibits green fluorescence and has a greater fluorescence increase upon protein binding, as compared with the BODIPY FL GTP-γ-S thioester. The BODIPY TR GTP-γ-S thioester is a red-fluorescent analog with spectral properties similar to the Texas Red dye.

We also offer the green-fluorescent BODIPY FL GTP-γ-NH amide (G35778, Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins - Section 17.3) as another choice for protein-binding studies. Although this analog exhibits less fluorescence enhancement upon protein binding, it is reportedly the best of the three green-fluorescent GTP-γ analogs for directly monitoring nucleotide exchange.ref The different linker lengths of the green-fluorescent GTP-γ analogs (six-carbon for BODIPY FL GTP-γ-NH amide, four- carbon for BODIPY FL GTP-γ-S and one-carbon for BODIPY 515/530 GTP-γ-S) may be useful for understanding protein active-site geometries.

In addition to their potential use for binding studies, our Patented BODIPY FL ATP-γ-S and BODIPY FL GTP-γ-S thioesters are important substrates for Fhit (Figure 17.18), a member of the histidine triad superfamily of nucleotide-binding proteins that bind and cleave diadenosine polyphosphates.ref Fhit, one of the most frequently inactivated proteins in lung cancer, functions as a tumor suppressor by inducing apoptosis.ref These BODIPY nucleotides should be especially useful for screening potential Fhit inhibitors and activators.




Figure 17.18 Principle of fluorescence-based detection of the diadenosine triphosphate hydrolase activity of Fhit using BODIPY FL GTP-γ-S thioester (G22183) as a substrate analog.


Conjugates of BODIPY Dyes for Fluorescence Polarization–Based Assays

The relatively long fluorescence lifetimes (typically >4 nanoseconds) at visible wavelengths, good anisotropy properties, high molar absorptivity and fluorescence intensity and lack of pH sensitivity in the spectra of the BODIPY dyes have been shown to make these dyes the preferred fluorophores for high-throughput, fluorescence polarization–based assays (Fluorescence Polarization (FP) - Note 1.5). BODIPY dye conjugates of nucleotides, peptides and drug analogs are available from Molecular Probes or are readily prepared from the chemically reactive BODIPY dyes. Fluorescence polarization–based assays for G-protein–coupled receptors, kinases and phosphatases and for high-affinity receptors are particularly important when screening for new drug candidates. Our EnzChek Polarization Assay Kit for Proteases (E6658, Detecting Peptidases and Proteases - Section 10.4, EnzChek(R) Polarization Assay Kit for Proteases), which contains green-fluorescent BODIPY FL casein with an optimal degree of labeling, is particularly useful for fluorescence polarization–based protease assays.

Additional Methods of Analysis Using BODIPY Dye Conjugates

In addition to their general utility for the intensity-based and fluorescence polarization–based assays described above, the BODIPY dyes are near optimal for a variety of other bioanalytical techniques:

  • The spectral variety and high absorbance of the BODIPY dyes (Figure 1.41) permits their use as efficient donor or acceptor dyes for numerous assays that use fluorescence resonance energy transfer, including internally quenched endopeptidase substrates ref (Detecting Peptidases and Proteases - Section 10.4), nucleic acid hybridization assays (Detecting Nucleic Acid Hybridization - Section 8.5) and receptor-binding assays (Fluorescence Resonance Energy Transfer (FRET) - Note 1.2 ).
  • BODIPY dye conjugates of peptides are readily separated by chromatographic means and can be used to detect the activity of enzymes that catalyze secondary modifications, such as phosphorylation/dephosphorylation, glycosylation/deglycosylation, oxidation/reduction, myristoylation, farnesylation and peptide–peptide crosslinking.
  • Hydrolysis of peptides that are singly labeled with BODIPY dyes to smaller peptides can be detected chromatographically with extremely high sensitivity.
  • In several instances, we have observed significant fluorescence enhancement or quenching of BODIPY dye–labeled probes on binding to receptors or in hybridization assays that may permit sensitive, high-throughput assays that do not require separation steps.
  • With their high peak intensity and narrow emission spectra, reactive BODIPY dyes are among the most detectable amine-derivatization reagents available for HPLC and capillary electrophoresis; thus, amine-containing metabolites can be derivatized with succinimidyl esters of the BODIPY dyes (Amine-reactive BODIPY dyes - Table 1.7) for ultrasensitive analysis.

Data Table

View Data Table Legend
Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
B10002 icon icon 648.31 F,D,L H2O, DMSO 541 60,000 569 MeCN 1, 2
B10006 icon icon 542.19 F,D,L H2O, DMSO 502 80,000 510 MeOH 1, 2
D2183 icon icon 292.09 F,L DMSO, MeCN 505 91,000 511 MeOH 1
D2184 icon icon 389.16 F,D,L DMSO, MeCN 502 82,000 510 MeOH 1, 3
D2187 icon 513.31 F,D,L DMSO, MeCN 534 77,000 551 MeOH 1
D2191 icon 417.22 F,D,L DMSO, MeCN 500 79,000 509 MeOH 1
D2219 icon 443.23 F,D,L DMSO, MeCN 559 97,000 568 MeOH 1
D2222 icon 463.25 F,D,L DMSO, MeCN 563 142,000 569 MeOH 1
D2225 icon 426.19 F,D,L DMSO, MeCN 575 83,000 588 MeOH 1
D2228 icon 489.28 F,D,L DMSO, MeCN 581 136,000 591 MeOH 4
D3834 icon icon 320.15 F,L DMSO, MeCN 505 96,000 511 MeOH 1
D6102 icon icon 502.32 F,D,L DMSO, MeCN 504 85,000 510 MeOH 1
D6116 icon icon 634.46 F,D,L DMSO, MeCN 588 68,000 616 MeOH 1, 5
D6117 icon icon 608.45 F,D,L DMSO, MeCN 544 60,000 570 MeOH 1
D6140 icon icon 491.20 F,D,L H2O, DMSO 502 75,000 510 MeOH 1, 6
D6180 icon icon 437.21 F,D,L DMSO, MeCN 528 70,000 547 MeOH 1
D6184 icon icon 417.22 F,D,L DMSO, MeCN 504 87,000 511 MeOH 1
D10000 icon icon 660.50 F,D,L DMSO, MeCN 625 101,000 640 MeOH 1, 7
D10001 icon icon 643.45 F,D,L DMSO, MeCN 646 102,000 660 MeOH 1

1. The absorption and fluorescence spectra of BODIPY derivatives are relatively insensitive to the solvent.
2. This sulfotetrafluorophenyl (STP) ester derivative is water-soluble and may be dissolved in buffer at ~pH 8 for reaction with amines. Long-term storage in water is NOT recommended due to hydrolysis.
3. The fluorescence lifetime (τ) of D2184 in MeOH at 20°C is 5.7 nanoseconds. Data provided by the SPEX Fluorescence Group, Jobin Yvon Inc.
4. Oxidation of the polyunsaturated butadienyl portion of the BODIPY 581/591 dye results in a shift of the fluorescence emission peak from ~590 nm to ~510 nm.ref
5. The fluorescence lifetime (τ) of D6116 in MeOH at 20°C is 5.4 nanoseconds. Data provided by the SPEX Fluorescence Group, Jobin Yvon Inc.
6. This sulfonated succinimidyl ester derivative is water-soluble and may be dissolved in buffer at ~pH 8 for reaction with amines. Long-term storage in water is NOT recommended due to hydrolysis.
7. The fluorescence lifetime (τ) of the BODIPY 630/650 dye at 20°C is 3.9 nanoseconds in H2O and 4.4 nanoseconds in EtOH.ref