The Molecular Probes Handbook describes a full spectrum of fluorophores and haptens for covalent derivatization of biopolymers and low molecular weight molecules. Chapters 1–5 describe the chemical and spectroscopic properties of the reactive reagents we offer, whereas the remainder of this book is primarily devoted to our diverse collection of fluorescent probes and their applications in cell biology, neurobiology, immunology, molecular biology and biophysics.

Common Applications for Amine-Reactive

Labeling Biomolecules

Amine-reactive probes are widely used to modify proteins, peptides, ligands, synthetic oligonucleotides and other biomolecules. In contrast to our thiol-reactive reagents (Thiol-Reactive Probes—Chapter 2), which frequently serve as probes of protein structure and function, amine-reactive dyes are most often used to prepare bioconjugates for immunochemistry, fluorescence in situ hybridization (FISH), cell tracing, receptor labeling and fluorescent analog cytochemistry. In these applications, the stability of the chemical bond between the dye and biomolecule is critical. The bioconjugate will typically be applied to a biochemically complex (and sometimes active) specimen. Furthermore, it will often be subjected to a series of post-processing steps such as washing, permeabilization, fixation and mounting. The integrity of the bioconjugate must be maintained throughout these processes if the fluorescence signal generated by the dye is to have any useful relationship to the abundance or localization of the bioconjugate's molecular target.

Our selection of amine-reactive fluorophores for modifying biomolecules covers the entire visible and near-infrared spectrum (Molecular Probes amine-reactive dyes—Table 1.1, Active esters and kits for labeling proteins and nucleic acids—Table 1.2). Up-to-date bibliographies are available for most of our amine-reactive probes at Also available are other product-specific bibliographies, as well as keyword searches of the over 60,000 literature references in our product application bibliography database.

Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 discusses the properties of Molecular Probes fluorophores, including:

Our essentially nonfluorescent QSY dyes (Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6, Reagents for Analysis of Low Molecular Weight Amines—Section 1.8) have strong visible absorption, making them excellent acceptors for fluorescence resonance energy transfer (FRET) applications (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).

Preparing the Optimal Bioconjugate

The preferred bioconjugate usually has a high fluorescence output (or, in the case of a haptenylated conjugate, a suitable degree of labeling) yet retains the critical functional properties of the unlabeled biomolecule, such as solubility, selective binding to a receptor or nucleic acid, activation or inhibition of a particular enzyme or the ability to incorporate into a biological membrane.ref Frequently, however, conjugates with the highest degree of labeling precipitate out of solution or bind nonspecifically.ref Thus there is usually a trade-off between degree of labeling and functional properties that must be resolved through experimental optimization. Lysine residues, the primary targets for amine modification of proteins, are relatively abundant. In mammalian proteins, lysine has the fifth highest occurrence frequency of the 20 naturally occurring amino acids. A typical IgG antibody molecule has about 90 lysine residues, of which about 30 at most can be modified under forcing conditions of high acylating reagent concentration and prolonged incubation. However, maintenance of functional properties (more specifically, antigen binding affinity) typically requires a degree of labeling of <10 dyes per IgG, representing a low fractional modification of available targets. A further consequence of low fractional modification is that, with the exception of small peptides or rare proteins with few lysine residues, bioconjugates prepared by amine modification are polydisperse mixtures containing a range of dye:protein stoichiometries.ref

For the most critical assays, we recommend that researchers consider preparing and optimizing their own conjugates. Our amine-reactive dyes are supplied with a detailed protocol that describes how to use them for labeling biomolecules. This procedure is straightforward and requires no special equipment. Following conjugation, it is very important to remove as much unconjugated labeling reagent as possible, usually by gel filtration, dialysis, bioconjugate precipitation and resolubilization, HPLC or a combination of these techniques. The presence of free dye, particularly if it remains chemically reactive, can greatly complicate subsequent experiments with the bioconjugate. The entire process of labeling reaction and conjugate purification can be completed in little more than two hours, and the main prerequisite is a sufficient amount of purified protein or amine-modified nucleic acid.

With the exception of the phycobiliproteins (Phycobiliproteins—Section 6.4, Spectral data for B-PE, R-PE and APC—Table 6.2), fluorescent microspheres (Microspheres—Section 6.5, FluoSpheres fluorescent microspheres—Table 6.7), Qdot nanocrystals (Qdot Nanocrystals—Section 6.6), Zenon Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.14) and ULYSIS Nucleic Acid Labeling Kits (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Molecular Probes nucleic acid labeling kits—Table 8.6), virtually all reagents used to prepare Molecular Probes fluorescent bioconjugates are amine-reactive organic fluorophores , and almost all are described in this chapter.

We have also developed convenient kit formats for labeling proteins and nucleic acids with our most important fluorophores, or alternatively with biotin. Kits for Labeling Proteins and Nucleic Acids—Section 1.2 and Molecular Probes kits for protein and nucleic acid labeling—Table 1.3 include a complete description of these kits. Alternatively, we prepare custom fluorescent conjugates for research use; contact Invitrogen Custom Services for more information. Conjugations with phycobiliproteins, fluorescent polystyrene microspheres, and Qdot nanocrystals require specialized procedures that are described in Phycobiliproteins—Section 6.4, Microspheres—Section 6.5 and Qdot Nanocrystals—Section 6.6, respectively.

Derivatizing Low Molecular Weight Molecules

Some amine-reactive probes described in this chapter are also important reagents for various bioanalytical applications, including amine quantitation, protein and nucleic acid sequencing and chromatographic and electrophoretic analysis of low molecular weight molecules. Reagents that are particularly useful for derivatizing low molecular weight amines—including fluorescamine, o-phthaldialdehyde, ATTO-TAG reagents, NBD chloride and dansyl chloride—are discussed in Reagents for Analysis of Low Molecular Weight Amines—Section 1.8. However, many of the reactive dyes described in Sections 1.2 to 1.7 can also be used as derivatization reagents; likewise, most of the derivatization reagents in Reagents for Analysis of Low Molecular Weight Amines—Section 1.8 can be utilized for biomolecule conjugation.

Reactivity of Amino Groups

The amine-reactive organic fluorophores described in this chapter are mostly acylating reagents that form carboxamides, sulfonamides or thioureas upon reaction with amines. The kinetics of the reaction depend on the reactivity and concentration of both the acylating reagent and the amine.ref Of course, buffers that contain free amines such as Tris and glycine must be avoided when using any amine-reactive probe. Ammonium sulfate used for protein precipitation must also be removed before performing dye conjugations. In addition, high concentrations of nucleophilic thiols should be avoided because they may react with the amine-reactive reagent to form an unstable intermediate that could consume the dye. Reagents for reductive alkylation of amines are described in Thiol-Reactive Probes—Chapter 2 and Click Chemistry and Other Functional Group Modifications—Chapter 3.

The most significant factors affecting an amine's reactivity are its class (aliphatic or aromatic) and its basicity. Virtually all proteins have lysine residues, and most have a free amine at the N-terminus. Aliphatic amines such as lysine's ε-amino group are moderately basic and reactive with most acylating reagents. However, the concentration of the free base form of aliphatic amines below pH 8 is very low; thus, the kinetics of amine acylation by isothiocyanates, succinimidyl esters or other reagents are strongly pH dependent. A pH of 8.5 to 9.5 is usually optimal for modifying lysine residues. In contrast, the α-amino group at a protein's N-terminus usually has a pKa of ~7, so it can sometimes be selectively modified by reaction at near neutral pH. Furthermore, although amine acylation should usually be carried out above pH 8.5, the acylation reagents tend to degrade in the presence of water, with the rate increasing as the pH increases.ref Protein modification by succinimidyl esters can typically be done at pH 8.3, whereas isothiocyanates usually require a pH >9 for optimal conjugations; this high pH may be a factor when working with base-sensitive proteins. DNA and most polysaccharides can be modified at a relatively basic pH if necessary.

Aromatic amines, which are uncommon in biomolecules, are very weak bases and thus unprotonated at pH 7. Modification of aromatic amines requires a highly reactive reagent, such as an isocyanate, isothiocyanate, sulfonyl chloride or acid halide, but can be done at any pH above ~4. A tyrosine residue can be selectively modified to form an o-aminotyrosine aromatic amine, which can then be reacted at a relatively low pH with certain amine-reactive probes ref (Figure 1.1.1).

In aqueous solution, acylating reagents are virtually unreactive with the amide group of peptide bonds and the side-chain amides of glutamine and asparagine residues, the guanidinium group of arginine, the imidazolium group of histidine and the nonbasic amines, such as adenosine or guanosine, found in nucleotides and nucleic acids.


Nitration of tyrosine by reaction with tetranitromethane

Figure 1.1.1.
Nitration of tyrosine by reaction with tetranitromethane, followed by reduction with sodium dithionite, to yield an o-aminotyrosine.



















Because they are very susceptible to deterioration during storage, we do not sell any isocyanates (R–NCO). Some acyl azides (Reagents for Modifying Alcohols—Section 3.2), however, are readily converted to isocyanates (Figure 1.1.2), which then react with amines to form ureas. As an alternative to the unstable isocyanates, we offer a large selection of isothiocyanates (R–NCS), which are moderately reactive but quite stable in water and most solvents. Isothiocyanates form thioureas upon reaction with amines (Figure 1.1.3). Although the thiourea product is reasonably stable, it has been reported that antibody conjugates prepared from fluorescent isothiocyanates deteriorate over time,ref prompting us to use fluorescent succinimidyl esters and sulfonyl halides almost exclusively for synthesizing bioconjugates. The thiourea formed by the reaction of fluorescein isothiocyanate with amines is also susceptible to conversion to a guanidine by concentrated ammonia.ref Despite the growing number of choices in amine-reactive fluorophores, fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) are still widely used reactive fluorescent dyes for preparing fluorescent bioconjugates.


Diacetate of fluorescein-5-carbonyl azide

Figure 1.1.2. Derivatization of an alcohol using the diacetate of fluorescein-5-carbonyl azide (F6218). This process consists of three steps: 1) rearrangement of the acyl azide to an isocyanate, 2) reaction of the isocyanate with an alcohol to form a urethane and 3) deprotection of the nonfluorescent urethane derivative using hydroxylamine.



Figure 1.1.3. Reaction of a primary amine with an isothiocyanate.

Active Esters and Carboxylic Acids

Succinimidyl Esters

Succinimidyl esters are reliable reagents for amine modification because the amide bonds they form (Figure 1.1.4) are as stable as peptide bonds. We provide over 100 succinimidyl esters of fluorescent dyes and nonfluorescent molecules, most of which have been developed within our own laboratories (Molecular Probes amine-reactive dyes—Table 1.1, Active esters and kits for labeling proteins and nucleic acids—Table 1.2). These reagents are generally stable during storage if well desiccated, and they show good reactivity with aliphatic amines and very low reactivity with aromatic amines, alcohols, phenols (including tyrosine) and histidine. Side-reactions of succinimidyl esters with alcohols are generally only observed in applications such as derivatization for mass spectrometry, in which much larger molar excesses of succinimidyl ester reagents and longer reaction times are used than is typically the case in protein labeling for fluorescence detection applications.ref Succinimidyl esters will also react with thiols in organic solvents to form thioesters. If formed in a protein, a thioester may transfer the acyl moiety to a nearby amine. Succinimidyl ester hydrolysis (generating the unreactive carboxylic acid) competes with conjugation, but this side reaction is usually slow below pH 9.

Tetrafluorophenyl (TFP) ester

Figure 1.1.4.
Reaction of a primary amine with a succinimidyl ester or a tetrafluorophenyl (TFP) ester.






Carboxylic Esters and Their Conversion into Sulfosuccinimidyl Esters and STP Esters

Some succinimidyl esters may not be compatible with a specific application because they can be quite insoluble in aqueous solution. To overcome this limitation, we offer carboxylic acid derivatives of many fluorophores, which can be converted into sulfosuccinimidyl esters or 4-sulfotetrafluorophenyl (STP) esters. These sulfonated esters have higher water solubility than simple succinimidyl esters and sometimes eliminate the need for organic solvents in the conjugation reaction. They are, however, also more polar than succinimidyl esters, which makes them less likely to react with buried amines in proteins or to penetrate cell membranes. Because of their combination of reactivity and polarity, sulfosuccinimidyl esters are not easily purified by chromatographic means and thus only a few are currently available. Sulfosuccinimidyl esters can generally be prepared in situ simply by dissolving the carboxylic acid dye in an amine-free buffer that contains N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHSS, H2249; EDAC, E2247; Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4). Addition of NHSS to the buffer has been shown to enhance the yield of carbodiimide-mediated conjugations ref (Figure 1.1.5). STP esters (Figure 1.1.6) are prepared in the same way from 4-sulfo-2,3,5,6-tetrafluorophenol ref (S10490, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4), and we find them to be more readily purified by chromatography than their sulfosuccinimidyl ester counterparts. The carboxylic acid derivatives may also be useful for preparing acid chlorides and anhydrides, which, unlike succinimidyl esters, can be used to modify aromatic amines and alcohols.


Figure 1.1.5. Stabilization of an unstable O-acylisourea intermediate by N-hydroxysulfosuccinimide (NHSS, H2249) in a carbodiimide-mediated (EDAC, E2247) modification of a carboxylic acid with a primary amine.

Primary amine with an STP ester

Figure 1.1.6. Reaction of a primary amine with an STP ester.

Tetrafluorophenyl (TFP) Esters

2,4,5,6-Tetrafluorophenyl (TFP) esters (Figure 1.1.4) are more resistant to nonspecific hydrolysis than either succinimidyl esters (Figure 1.1.7) or sulfosuccinimidyl esters, yet they exhibit equal or superior reactivity with amines.ref At this time, Alexa Fluor 488 carboxylic acid is the only fluorescent TFP ester we offer (A30005, Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3).

Succinimidyl (NHS) esters at basic pH

Figure 1.1.7.
Stability of the tetrafluorophenyl (TFP) and succinimidyl (NHS) esters at basic pH (8.0–9.0).








Sulfodichlorophenol (SDP) Esters

The sulfodicholorphenol (SDP) ester is currently the most hydrolytically stable amine-reactive moiety that we offer. As with TFP esters, Alexa Fluor 488 carboxylic acid is the only fluorescent SDP ester available (A30052, Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3). Conjugates produced with the Alexa Fluor 488 5-SDP ester produce the same strong amide bond between the dye and the compound of interest as succinimidyl and tetrafluorophenyl (TFP) esters. Because of its improved stability in water and buffers, however, the SDP ester can potentially offer increased control and consistency in reactions as compared with its succinimidyl ester and TFP ester counterparts.

Carbonyl Azides

Reagents for Modifying Alcohols—Section 3.2 describes coumarin, fluorescein and tetramethylrhodamine carbonyl azides (D1446, M1445, F6218, T6219). Like succinimidyl esters, carbonyl azides are active esters that can react with amines to yield amides; however, a more common application of carbonyl azides is thermal rearrangement to a labile isocyanate (which can then react with both aliphatic and aromatic amines to form ureas) for derivatizing alcohols and phenols (Reagents for Modifying Alcohols—Section 3.2, Figure 1.1.2). 

Sulfonyl Chlorides

Sulfonyl chlorides, including the dansyl, pyrene, Lissamine rhodamine B and Texas Red derivatives, are highly reactive but also quite unstable in water, especially at the higher pH required for reaction with aliphatic amines. For example, we have determined that dilute Texas Red sulfonyl chloride is totally hydrolyzed within 2–3 minutes in pH 8.3 aqueous solution at room temperature.ref Protein modification by this reagent is therefore best done at low temperature. Once conjugated, however, the sulfonamides that are formed (Figure 1.1.8) are extremely stable; they even survive complete protein hydrolysis (for example, dansyl end-group analysis ref).

Sulfonyl chlorides can also react with phenols (including tyrosine), aliphatic alcohols (including polysaccharides), thiols (such as cysteine) and imidazoles (such as histidine), but these reactions are not common in proteins or in aqueous solution. Sulfonyl chloride conjugates of thiols and imidazoles are generally unstable, and conjugates of aliphatic alcohols are subject to nucleophilic displacement.ref Note that sulfonyl chlorides are unstable in dimethylsulfoxide (DMSO) and should never be used in that solvent.ref

Sulfonyl chloride

Figure 1.1.8. Reaction of a primary amine with a sulfonyl chloride.

Other Amine-Reactive Reagents

Aldehydes react with amines to form Schiff bases. Notable aldehyde-containing reagents described in Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7 include o-phthaldialdehyde (OPA) and naphthalenedicarboxaldehyde (NDA), as well as the 3-acylquinolinecarboxaldehyde (ATTO-TAG) reagents CBQCA and FQ devised by Novotny and collaborators.ref All of these reagents are useful for the sensitive quantitation of amines in solution, by HPLC and by capillary electrophoresis. In addition, certain arylating reagents such as NBD chloride, NBD fluoride and dichlorotriazines react with both amines and thiols, forming bonds with amines that are particularly stable.

For Research Use Only. Not for use in diagnostic procedures.