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Amine Reactive Dyes and Probes for Conjugation

Amine-reactive probes, composed of an amine-reactive moiety and a chemical moiety or protein, are widely used in bioconjugation for their convenience and performance. The strength and stability of the chemical bond between tag and biomolecule produces robust bioconjugates resistant to the harsh conditions of post-processing steps synonymous with applications such as immunohistochemistry (IHC), immunocytochemistry (ICC), flow cytometry, enzyme-linked immunosorbent assays (ELISA), fluorescence in situ hybridization (FISH), cell tracing, and receptor labeling. AAT Bioquest offers a wide variety of amine-reactive probes for labeling proteins, peptides, amine-modified oligonucleotides, and other biomolecules with fluorophores, enzymes, or hapten tags. Featured in this portfolio are our bright and photostable iFluor™ dyes that span the entire UV, visible, and IR spectrum, providing researchers with more possibilities for multiplex detection.

Too busy to perform your conjugation or having difficulty finding the right amine-reactive probe. Not to worry, AAT Bioquest's bioconjugation and custom oligonucleotide labeling services can prepare the bioconjugate you need.

 

 

 

Common Amine-Reactive Moieties and Their Chemical Reactivity


Primary amines (-NH2) are mainly found at the N-terminus of polypeptide chains (i.e., α-amine) and on the side chains of lysine residues (i.e., the ε-amine). Compared to other conjugation targets, such as sulfhydryls (-SH), primary amines have an outward-facing orientation at physiological conditions making them readily accessible for conjugation. When conjugating with amine-reactive probes, it is important to avoid using buffers containing free amines, such as Tris and glycine, as they will compete with the labeling reaction. In addition, any inorganic salts, such as ammonium sulfate, and stabilizing reagents, such as bovine serum albumin (BSA), must be removed via dialysis or some other purification method before labeling. Failure to do so can significantly impact conjugation yield.

While several types of amine-reactive moieties exist, the most commonly used include succinimidyl esters, imidioesters, sulfonyl chlorides, and isothiocyanates. These acylating reagents react with nucleophiles to form acylated products, and as a result, the reactive moiety is displaced. Two key factors to consider when selecting an amine-reactive probe are the reactivity class of the amine group - aliphatic or aromatic - and its basicity. Aliphatic amines, which are located on the ε-amino group of lysine residues, exist on most proteins. They are moderately basic and will react with most acylating reagents optimally at a pH of 8.0 to 9.5. Aromatic amines, however, are not so common. These weak bases are unprotonated at a neutral pH and typically require a highly reactive moiety, such as an isothiocyanate or sulfonly chloride.

Succinimidyl Esters



Figure 1. The reaction of a succinimidyl ester compound with a primary amine.
Succinimidyl ester (SE or NHS esters) derivatives are among the most popular conjugation reagents for amine modification. They are relatively easy to prepare, stable for storage, functional in an aqueous environment, and highly reactive for aliphatic amines versus aromatic amines, alcohols, phenols, and histidines. In SE conjugation, the carbonyl carbon of the SE moiety is attacked by a primary aliphatic amine (i.e., nucleophilic acyl substitution), resulting in a tetrahedral intermediate and the displacement of the SE group (i.e., leaving group). The nucleophile takes the place of the leaving group, and a stable amide bond is formed.

SE conjugation is best performed under physiological to slightly alkaline conditions (pH 7-9) with phosphate, carbonate-bicarbonate, HEPES, or borate buffers. Avoid buffers containing primary amines, such as Tris or glycine buffers. These buffers are incompatible with SE reagents and will compete for reaction decreasing conjugation yield. At pH levels closer to 9, the reaction efficiency can improve due to a higher degree of amine deprotonation; however, the rate of SE hydrolysis will also increase. For less concentrated protein solutions, this can result in less-efficient crosslinking.

AAT Bioquest offers a wide selection of SE reagents, including fluorescent and non-fluorescent dye derivatives. These include iFluor® dyes, Alexa Fluor® dye equivalents, conventional fluorophores, non-fluorescent quenchers, pH-sensitive dyes, calcium indicator Cal-520®, biotin, digoxigenin (DIG), DNP, horseradish peroxidase (HRP), estradiol, fentanyl, and more (see table 1 below).
 

Table 1. Fluorophore, hapten, enzyme and other succinimidyl ester derivatives for labeling proteins, antibodies and peptides.

Product
Ex (nm)
Em (nm)
ε¹
φ²
CF at 280³
Unit Size
Cat No.
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*493517830000.17825 mg110
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*493517830000.178100 mg111
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*493517830000.1781 g112
5-FAM, SE [5-Carboxyfluorescein, succinimidyl ester] *CAS 92557-80-7*493517830000.17810 mg113
5-FAM, SE [5-Carboxyfluorescein, succinimidyl ester] *CAS 92557-80-7*493517830000.178100 mg114
5-FAM, SE [5-Carboxyfluorescein, succinimidyl ester] *CAS 92557-80-7*493517830000.1781 g115
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*493517830000.17810 mg116
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*493517830000.178100 mg117
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*493517830000.1781 g118
Cyanine 3 bissuccinimidyl ester [equivalent to Cy3® bisNHS ester]5555691500000.150.0731 mg138
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. φ = fluorescence quantum yield.
  3. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).


Sulfonyl Chlorides



Figure 2. The reaction of a sulfonyl chloride compound with a primary amine.
Sulfonyl chlorides are highly reactive compounds for amine modification. Reaction of a sulfonyl chloride reagent with a protein or other amine-containing molecule results in the loss of the chlorine atom and the formation of a very stable sulfonamide bond. Although the stability of sulfonamide bonds are strong enough to withstand complete protein hydrolysis, sulfonyl chlorides are much more challenging to use and, therefore, not recommended for routine protein labeling. Conjugation occurs optimally under alkaline conditions (pH 9-10) in phosphate, bicarbonate, and borate buffers. Sulfonyl chlorides are quite unstable in aqueous solutions, especially at higher pH levels, and should never be used with the solvent dimethylsulfoxide (DMSO), as it reacts with them.
 

Table 2. Fluorescent dye sulfonyl chlorides for labeling proteins, antibodies and peptides.

Product
Ex (nm)
Em (nm)
ε¹
φ²
CF at 280³
Unit Size
Cat No.
Lissamine Rhodamine B Sulfonyl Chloride [Sulforhodamine B sulfonyl chloride] *CAS 62796-29-6*558575850000.330.171100 mg470
Sulforhodamine 101 sulfonyl chloride [Texas Red®] *CAS 82354-19-6*5866031160000.930.3610 mg480
Dansyl chloride [5-Dimethylaminonaphthalene-1-sulfonyl chloride] *CAS 605-65-2*33551833000.700.387100 mg811
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. φ = fluorescence quantum yield.
  3. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

Isothiocyanates



Figure 3. The reaction of an isothiocyanate compound with a primary amine.
Isothiocyanates are moderately reactive electrophilic compounds that react with amino, hydroxyl, or thiol groups to form thioureas, O-thiocarbamates, or dithiocarbamates. In the reaction, the central, electrophilic carbon of the isothiocyanate moiety is attacked by an amine nucleophile. The resulting electron shift and proton loss create a thiourea bond between the isothiocyanate reagent and amine-containing compound with no leaving group. For isothiocyanate compounds, conjugation occurs optimally at alkaline pH levels (pH > 9), where the target amino groups are unprotonated (aromatic amines). Compared to bioconjugates modified with succinimidyl esters or sulfonyl chlorides, thiourea products are less stable and are more susceptible to deterioration over time. Popular isothiocyanate derivatives include fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) for labeling proteins, particularly antibodies, with green or orange/red fluorescence, respectively.
 

Table 3. Fluorescent dye isothiocyanates for labeling proteins, antibodies, and peptides.

Product
Ex (nm)
Em (nm)
ε¹
Φ²
CF at 280 nm³
Unit Size
Cat No.
5-FITC [Fluorescein-5-isothiocyanate] *CAS 3326-32-7*491516730000.920.254100 mg120
5-FITC [Fluorescein-5-isothiocyanate] *CAS 3326-32-7*491516730000.920.2541 g121
5-FITC [Fluorescein-5-isothiocyanate] *CAS 3326-32-7*491516730000.920.25410 g122
5(6)-TRITC [Tetramethylrhodamine-5-(and-6)-isothiocyanate] *CAS 95197-95-8*5445701000000.100.3425 mg409
5(6)-TRITC [Tetramethylrhodamine-5-(and-6)-isothiocyanate] *CAS 95197-95-8*5445701000000.100.345 mg410
5-TRITC [Tetramethylrhodamine-5-isothiocyanate] *CAS 80724-19-2*5445701000000.100.341 mg415
5-TRITC [Tetramethylrhodamine-5-isothiocyanate] *CAS 80724-19-2*5445701000000.100.345 mg416
6-TRITC [Tetramethylrhodamine-6-isothiocyanate] *CAS 80724-20-5*5445701000000.100.341 mg417
6-TRITC [Tetramethylrhodamine-6-isothiocyanate] *CAS 80724-20-5*5445701000000.100.345 mg418
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. φ = fluorescence quantum yield.
  3. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

 

Optimal Bioconjugation with Amine-Reactive Fluorophores


Qualities of a desirable bioconjugate include one that is highly fluorescent, less susceptible to photobleaching and exhibits good water solubility while retaining the same functional properties as its innate unmodified counterpart. Two factors that contribute considerably to achieving these qualities include the fluorophore itself and the number of dyes attached to the protein, more commonly referred to as the degree of labeling (DOL).

Fluorophores with high extinction coefficients and high quantum yields will generate bright fluorescence signals upon excitation, and the higher these values are, the brighter the fluorophore. For instance, phycoerythrin (PE), which has an extinction coefficient of 1,960,000 cm-1M-1, is one of the brightest fluorescent dyes known and is widely used to image low-abundance cellular targets in flow cytometry. However, due to its rapid photobleaching, PE has limited use in immunofluorescence microscopy. FITC, another widely used labeling dye for biological research, benefits from high absorptivity, excellent fluorescent quantum yield, and good water solubility, but its high rate of photobleaching and pH-sensitive fluorescence can affect its performance, especially for staining protocols using acidic buffers. New generation dyes, such as iFluor® dyes and Alexa Fluor® dyes, are highly photostable and maintain high fluorescence intensities over a broad pH range. The enhanced photostability and high quantum yield of iFluor® 488 (φ=0.9) enable the detection of biological targets with greater sensitivity.

More important than selecting the right fluorophore for any experiment is optimizing the degree of labeling such that the bioconjugate retains its functionality and has a high fluorescence output. Since proteins contain numerous primary targets for amine modification, the amount of dye-labeled to a single protein can vary significantly. Conjugates with a low DOL may result in weak fluorescence, while a high DOL can cause conjugates to precipitate out of solution, bind nonspecifically, lose functionality, and self-quench. For antibodies, the optimal DOL usually ranges between 2 to 10. A more precise value, however, will largely depend on the properties of the label and protein. This means that for many bioconjugations, the optimal DOL must be experimentally determined, often through several small-batch labelings. For accurate determination of dye:protein molar ratios, use AAT Bioquest's DOL calculator

After conjugation, removing as much reactive-free dye as possible is essential. This can be done via gel filtration chromatography, dialysis, or using a desalting column, such as the ReadiUse™ Disposable PD-10 desalting column.


Fluorescence Spectrum Viewer

Need assistance selecting the best fluorophore for your experiment, use our Fluorescence Spectrum Viewer:

  • View and compare fluorophores and fluorescent proteins for biological applications
  • Check spectral compatibility
  • Add multiple excitation and emission filters
  • Save spectra configuration as a PNG or hyperlink