The thiol-reactive Alexa Fluor, BODIPY, fluorescein, Oregon Green, tetramethylrhodamine and Texas Red derivatives have strong absorptivity and high fluorescence quantum yields. This combination of attributes makes these compounds the preferred reagents for preparing protein and low molecular weight ligand conjugates to study the diffusion, structural properties and interactions of proteins and ligands using techniques such as:

In this section and in Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3, thiol-reactive reagents with similar spectra, rather than the same reactive group, are generally discussed together. The probes described in this section have visible absorption maxima beyond 410 nm; thiol-reactive probes with peak absorption below 410 nm are described in Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3. Thiol-reactive dyes excited with visible light—Table 2.1 summarizes this section's thiol-reactive probes excited with visible light.

Alexa Fluor Maleimides

Alexa Fluor dyes set new standards for fluorescent dyes and the bioconjugates prepared from them (The Alexa Fluor Dye Series—Note 1.1). Alexa Fluor dyes exhibit several unique features:

  • Strong absorption, with extinction coefficients greater than 65,000 cm-1M-1
  • Excellent photostability (Figure 2.2.1, Figure 2.2.2), providing more time for observation and image capture than spectrally similar dyes allow (photo)
  • pH-insensitive fluorescence between pH 4 and pH 10
  • Superior fluorescence output per protein conjugate, surpassing that of other spectrally similar fluorophore-labeled protein, including fluorescein, tetramethylrhodamine and Texas Red conjugates, as well as Cy3 and Cy5 conjugatesref

For labeling thiol groups, we offer thiol-reactive Alexa Fluor dyes that span the visible spectrum:

The Alexa Fluor maleimides are particularly useful for labeling thiol-containing proteins on the surface of live cells, where their polarity permits the sensitive detection of exposed thiols.ref In proteomics applications, Alexa Fluor protein conjugates can be electrophoretically separated and then detected without additional staining.ref As with their amine-reactive succinimidyl ester counterparts (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3), Alexa Fluor 647 maleimide, Alexa Fluor 750 maleimide and other long-wavelength reactive dyes are frequently used to make conjugates for in vivo imaging applications.ref In experiments using Alexa Fluor 488 maleimide, immunodetection of labeled proteins can be accomplished using our anti–Alexa Fluor 488 antibody (A11094, Anti-Dye and Anti-Hapten Antibodies—Section 7.4).

  Figure 2.2.1 Photobleaching resistance of the green-fluorescent Alexa Fluor 488, Oregon Green 488 and fluorescein dyes, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 488 (S11223, S32354), Oregon Green 488 (S6368) or fluorescein (S869) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). The cells were then fixed in 1% formaldehyde, washed and wet-mounted. After mounting, cells were scanned 10 times on a laser-scanning cytometer; laser power levels were 25 mW for the 488 nm spectral line of the argon-ion laser. Scan durations were approximately 5 minutes, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.
  Figure 2.2.2 Photobleaching resistance of the red-fluorescent Alexa Fluor 647, Alexa Fluor 633, PBXL-3 and Cy5 dyes and the allophycocyanin fluorescent protein, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 647 (S21374, S32357), Alexa Fluor 633 (S21375), PBXL-3, Cy5 or allophycocyanin (APC, S868) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). The cells were then fixed in 1% formaldehyde, washed and wet-mounted. After mounting, cells were scanned eight times on a laser-scanning cytometer; laser power levels were 18 mW for the 633 nm spectral line of the He-Ne laser. Scan durations were approximately 5 minutes, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.

BODIPY Derivatives

BODIPY Iodoacetamides, Maleimides and Methyl Bromides

Like their amine-reactive BODIPY counterparts (BODIPY Dye Series—Section 1.4), BODIPY iodoacetamides, BODIPY maleimides and BODIPY methyl bromides yield thiol adducts with several important properties:

  • High extinction coefficients (EC >60,000 cm-1M-1)
  • High fluorescence quantum yields, often approaching 1.0, even in water
  • Narrow emission bandwidths (Figure 2.2.3)
  • Good photostability ref
  • Spectra that are insensitive to pH and relatively insensitive to solvent polarity ref
  • Lack of ionic charge, which is especially useful when preparing membrane probes and cell-permeant reagents

BODIPY dyes are chemically stable between about pH 3 and pH 10, although they are less stable to extremes of pH than are fluorescein and Alexa Fluor derivatives. All of the thiol-reactive BODIPY dyes are suitable for labeling cysteine residues in proteins and thiolated oligonucleotides and for detecting thiol conjugates separated by HPLC and capillary electrophoresis using ultrasensitive laser-scanning techniques.ref BODIPY FL iodoacetamide has been shown to be highly selective for cysteine labeling, producing little or no nonspecific labeling even at high dye:thiol ratios; in contrast, tetramethylrhodamine iodoacetamide exhibited nonspecific labeling as dye concentrations increased.ref Furthermore, actin labeling with BODIPY FL iodoacetamide (D6003) reportedly does not perturb actin polymerization.ref BODIPY FL maleimide is a useful reagent for flow cytometric quantitation and confocal imaging of microparticles released upon agonist-elicited activation of human platelets.ref Labeling can be carried out after activation, avoiding concerns that pre-labeling might interfere with cellular functions involved in the activation process.

Our selection of thiol-reactive BODIPY reagents includes:

  • BODIPY FL maleimide and BODIPY FL iodoacetamide (B10250, Figure 2.2.4; D6003), which exhibit spectral characteristics very similar to fluorescein
  • BODIPY 507/545 iodoacetamide (D6004)
  • BODIPY TMR maleimide (B30466)
  • BODIPY 493/503 methyl bromide (B2103)
  • BODIPY 630/650 methyl bromide (B22802), with very long-wavelength spectra

Two additional symmetric maleimidylphenyl BODIPY derivatives are available with excitation/emission maxima of ~499/508 nm (D20350, Figure 2.2.4) and ~577/618 nm (D20351).

Figure 2.2.3
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 2.2.4
Comparison of the fluorophore orientation relative to the reactive moiety of two spectrally similar thiol-reactive BODIPY dyes: A) BODIPY 499/508 maleimide (D20350) and B) BODIPY FL N-(2-aminoethyl)maleimide (B10250).


We have attached the BODIPY FL fluorophore to the amino groups of the disulfide-linked amino acid cystine to create a reagent for reversible, thiol-specific labeling of proteins, thiolated oligonucleotides and cells.ref BODIPY FL L-cystine (B20340) is virtually nonfluorescent due to interactions between the two fluorophores; however, thiol-specific exchange to form a mixed disulfide results in significant enhancement of the green fluorescence (Figure 2.2.5).

Intramolecularly quenched BODIPY  
Figure 2.2.5
Reaction of intramolecularly quenched BODIPY FL L-cystine (B20340) with a thiol, yielding two fluorescent products—a mixed disulfide labeled with the BODIPY FL dye and a BODIPY FL cysteine derivative.

TS-Link BODIPY Thiosulfate Reagents

The TS-Link BODIPY reagents are water-soluble, fluorescent thiosulfates that react readily and selectively with free thiols to form disulfide bonds (Figure 2.2.6). In contrast to the thioether bonds formed by maleimides and iodoacetamides, the disulfide bond formed by the TS-Link reagents is reversible; the TS-Link BODIPY fluorophore can easily be removed using a reducing agent such as dithiothreitol or tris-(2-carboxyethyl)phosphine (DTT, D1532; TCEP, T2556; Introduction to Thiol Modification and Detection—Section 2.1), leaving the molecule of interest unchanged for downstream processing. These TS-Link reagents yield the same disulfide products as methanethiosulfonates (MTS reagents), but they are much more polar and water soluble and may therefore selectively react with residues on the surface of a protein or live cell.ref

We currently offer:

  • TS-Link BODIPY FL C2-thiosulfate (T30453)
  • TS-Link BODIPY TMR C5-thiosulfate (T30454)
  • TS-Link BODIPY TR C5-thiosulfate (T30455)
  • TS-Link BODIPY 630/650 C5-thiosulfate (T30456)

We also offer TS-Link DSB-X biotin C5-thiosulfate (TS-Link desthiobiotin-X C5-thiosulfate, T30754), which is described in Biotinylation and Haptenylation Reagents—Section 4.2.

Figure 2.2.6
Reaction of a TS-Link reagent (R1) with a thiol (R2), followed by removal of the label with a reducing agent.

Fluorescein Derivatives, Including Thiol-Reactive Oregon Green Dyes

Fluorescein Iodoacetamide, Maleimide and Methyl Bromide

The excellent water solubility of the fluorescein iodoacetamide single isomers (I30451, I30452) and fluorescein-5-maleimide (F150, structure) at pH 7 makes it easy to prepare green-fluorescent thiol conjugates of biomolecules. Fluorescein maleimide and 5-iodoacetamidofluorescein have been the most extensively used visible wavelength–excitable, thiol-reactive dyes for modifying proteins, nucleic acids and other biomolecules. Following conjugation to thiols, fluorescein-5-maleimide (and other fluoresceins) can be radioiodinated.ref

When compared with these iodoacetamide and maleimide derivatives, 5-(bromomethyl)fluorescein (B1355, structure) reacts more slowly with thiols of peptides, proteins and thiolated nucleic acids but forms stronger thioether bonds that are expected to remain stable under the conditions required for complete amino acid analysis. With the possible exception of our Alexa Fluor maleimides and the thiol-reactive BODIPY dyes described above, 5-(bromomethyl)fluorescein has the highest intrinsic detectability of all thiol-reactive probes, particularly for capillary electrophoresis instrumentation that uses the 488 nm spectral line of the argon-ion laser.ref

Oregon Green 488 Iodoacetamide and Maleimide

The Oregon Green 488 dye (2',7'-difluorofluorescein, D6145; Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) has absorption and emission spectra that are a perfect match to those of fluorescein. In addition to Oregon Green 488 isothiocyanate, carboxylic acid and succinimidyl ester derivatives (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5), we have synthesized the isomeric mixture of Oregon Green 488 iodoacetamide (O6010) and the single-isomer Oregon Green 488 maleimide (O6034, structure). These thiol-reactive probes yield conjugates that have several important advantages when directly compared with fluorescein conjugates, including:

  • Greater photostability (Figure 2.2.7)
  • A lower pKa (pKa of 4.8 for 2',7'-difluorofluorescein versus 6.4 for fluorescein) (Figure 2.2.8)
  • Higher fluorescence and less quenching at comparable degrees of substitution (Figure 2.2.9)
  • Utility as fluorescence anisotropy probes for measuring protein–protein and protein–nucleic acid interactions ref (Fluorescence Polarization (FP)—Note 1.4)
Figure 2.2.7
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.
Figure 2.2.8
Comparison of pH-dependent fluorescence of the Oregon Green 488 (filled circle), carboxyfluorescein (open circle) and Alexa Fluor 488 (open square) fluorophores. Fluorescence intensities were measured for equal concentrations of the three dyes using excitation/emission at 490/520 nm.
Figure 2.2.9
Comparison of relative fluorescence as a function of the number of fluorophores attached per protein for goat anti–mouse IgG antibody conjugates prepared using Oregon Green 514 carboxylic acid succinimidyl ester (O6139, filled square), Oregon Green 488 carboxylic acid succinimidyl ester (O6147, filled circle), fluorescein-5-EX succinimidyl ester (F6130, open circle) and fluorescein isothiocyanate (FITC, F143, F1906, F1907, open square). Conjugate fluorescence is 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.

Eosin Maleimide

As compared with the corresponding fluorescein derivative, eosin maleimide (E118, structure) is less fluorescent but much more phosphorescent and a better photosensitizer.ref With eosin's high quantum yield of 0.57 for singlet oxygen generation,ref eosin conjugates can be used as effective photooxidizers of diaminobenzidine (DAB) in high-resolution electron microscopy studies ref (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2).

Eosin (excitation/emission maxima ~519/540 nm) derivatives efficiently absorb the fluorescence from fluorescein and other fluorophores such as the BODIPY FL, Alexa Fluor 488, Oregon Green 488, dansyl and coumarin dyes, making them good acceptors in FRET techniques ref (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).

Although usually selectively reactive with thiols, eosin maleimide reportedly also reacts with a specific lysine residue of the band-3 protein in human erythrocytes, inhibiting anion exchange in these cells.ref A flow cytometry assay for hereditary spherocytosis (HS), characterized by band-3 protein–deficient erythrocytes, has been developed using this selective binding by eosin maleimide;ref in this assay, HS erythrocytes are identified as the population exhibiting low eosin fluorescence.

Rhodamine Derivatives, Including Thiol-Reactive Texas Red Dyes

Tetramethylrhodamine Iodoacetamide and Maleimide

Tetramethylrhodamine iodoacetamide (TMRIA) and tetramethylrhodamine maleimide yield photostable, pH-insensitive, red-orange–fluorescent thiol conjugates.ref These iodoacetamide and maleimide derivatives, however, are difficult to prepare in pure form and different batches of our mixed-isomer products have contained variable mixtures of the 5- and 6-isomers. Moreover, certain cytoskeletal proteins preferentially react with individual isomers, leading to complications in the interpretation of labeling results.ref Consequently, we now prepare the 5-isomer of TMRIA (T6006, structure) and the 5-isomer (T6027, structure) and 6-isomer (T6028, structure) of tetramethylrhodamine maleimide. A fluorogenic ADP biosensor has been described that exploits nucleotide-modulated self-quenching of two TMRIA labels that have been site-specifically attached to Escherichia coli ParM nucleotide-binding protein.ref Tetramethylrhodamine-5-maleimide is often used for voltage-clamp fluorometry,ref wherein it is attached to cysteine residues in the voltage-sensor domains of ion channels, generating fluorescence signals that are responsive to structural rearrangements associated with channel gating.ref In this context, the dye is sometimes referred to as TMRM, but it should not be confused with tetramethylrhodamine methyl ester (T668, Probes for Mitochondria—Section 12.2), a structurally similar but functionally quite different dye that is identified by the same acronym.

Rhodamine-Based Crosslinking Reagent

The thiol-reactive, homobifunctional crosslinker bis-((N-iodoacetyl)piperazinyl)sulfonerhodamine (B10621, structure) is derived from a relatively rigid rhodamine dye. It is similar to a thiol-reactive rhodamine-based crosslinking reagent used to label regulatory light-chains of chicken gizzard myosin for fluorescence polarization experiments.ref Researchers have attached bis-((N-iodoacetyl)piperazinyl)sulfonerhodamine to the kinesin motor domain and determined the orientation of kinesin bound to microtubules in the presence of a nonhydrolyzable ATP analog by fluorescence polarization microscopy.ref Images of single molecules of chicken calmodulin crosslinked between two engineered cysteines by bis-((N-iodoacetyl)piperazinyl)sulfonerhodamine have been used to generate comparisons of experimental and theoretical super-resolution point-spread functions ref (PSF). Dibromobimane (D1379, Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3) is a shorter-wavelength alternative for applications requiring a fluorescent homobifunctional thiol crosslinker.

Rhodamine Red Maleimide

We offer a maleimide derivative of our Rhodamine Red fluorophore (R6029), which is spectrally similar to Lissamine rhodamine B (Figure 2.2.10). The spectral properties of Rhodamine Red maleimide have been exploited to improve the light-harvesting efficiency of chlorophyll by site-specific labeling of cysteine residues in the recombinantly expressed apoprotein in order to fill in the "green gap" in the absorption spectrum.ref Rhodamine Red C2-maleimide is a mixture of two isomeric sulfonamides (structure).

Texas Red Bromoacetamide and Maleimide

Conjugates of the bromoacetamide and maleimide derivatives of our Texas Red fluorophore (T6009, T6008) have very little spectral overlap with fluorescein or Alexa Fluor 488 conjugates (Figure 2.2.10) and are therefore useful as second labels in multicolor applications or as energy transfer acceptors from green-fluorescent dyes.ref Bromoacetamides are only slightly less reactive with thiols than are iodoacetamides. The Texas Red bromoacetamide (structure) and maleimide (structure) derivatives are mixtures of the corresponding two isomeric sulfonamides.

Figure 2.2.10
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.

PyMPO Maleimide

PyMPO maleimide (M6026, structure) is an environment-sensitive thiol-reactive dye with a fluorescence excitation peak near 415 nm and an unusually long Stokes shift (fluorescence emission peak at ~560–580 nm). Its most widespread application is for labeling cysteine residues in the voltage-sensor domains of ion channels, where its fluorescence is exquisitely sensitive to structural rearrangements associated with channel gating.ref This technique is commonly referred to as voltage-clamp fluorometry.ref

Benzoxadiazole Derivatives, Including NBD Probes

NBD Chloride and NBD Fluoride

NBD chloride (C20260, structure) and the more reactive NBD fluoride (F486) are common reagents for amine modification (Reagents for Analysis of Low Molecular Weight Amines—Section 1.8). They also react with thiols ref and cysteine in several proteins ref to yield thioethers. NBD conjugates of thiols usually have much shorter-wavelength absorption and weaker fluorescence than do NBD conjugates of amines.ref Selective modification of cysteines in the presence of reactive lysines and tyrosines is promoted by carrying out the reaction at pH <7;ref however, NBD conjugates of thiols are often unstable, resulting in time-dependent label migration to adjacent lysine residues.ref


Thiol conjugates of 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide ref (ABD-F, F6053; structure) are much more stable in aqueous solution than are the thiol conjugates prepared from NBD chloride or NBD fluoride.ref ABD-F is nonfluorescent until reacted with thiols and therefore can be used to quantitate thiols in solution,ref as well as thiols separated by HPLC ref or TLC.ref ABD-F also reportedly reacts slowly with the hydroxy group of some tyrosine residues as well as α-amino groups in some proteins, forming products that are nonfluorescent but can be detected by absorbance at 385 nm.ref ABD-F labeling is blocked by zinc binding to protein thiols and can therefore be used as an inverse proportionality indicator of bound Zn2+.ref In contrast, the fluorescent zinc indicators described in Fluorescent Indicators for Zn2+ and Other Metal Ions—Section 19.7 primarily detect free Zn2+ ions. ABD–cysteine conjugates are very stable to acid hydrolysis, but labeling is partially reversed in basic solution containing DTT ref (D1532; Introduction to Thiol Modification and Detection—Section 2.1).

IANBD Ester and IANBD Amide

When conjugating the NBD fluorophore to thiols located in hydrophobic sites of proteins, we recommend using the NBD iodoacetate ester (IANBD ester, I9; structure) or, preferably, the more hydrolytically stable NBD iodoacetamide (IANBD amide, D2004; structure). These reactive reagents exhibit appreciable fluorescence only after reaction with thiols that are buried or unsolvated, and this fluorescence is highly sensitive to changes in protein conformation and assembly of molecular complexes.ref

Lucifer Yellow Iodoacetamide

Lucifer yellow CH is a well-known polar tracer for neurons (Polar Tracers—Section 14.3). Its iodoacetamide derivative (L1338, structure) similarly has high water solubility and visible absorption and emission spectra similar to those of lucifer yellow CH (spectra). As with the polar Alexa Fluor maleimides (see above) and the stilbene iodoacetamide and maleimide (A484, A485; Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3), a principal application of lucifer yellow iodoacetamide is the labeling of exposed thiols of proteins in solution, as well as in the outer membrane of live cells.ref Lucifer yellow iodoacetamide has also been used as a fluorescence energy acceptor from aequorin in bioluminescence resonance energy transfer (BRET) assays.ref

TC-FlAsH and TC-ReAsH Detection of Tetracysteine-Tagged Proteins

TC-FlAsH and TC-ReAsH Detection Technology

TC-FlAsH and TC-ReAsH detection technology, based on the tetracysteine tag first described by Griffin, Adams and Tsien in 1998,ref takes advantage of the high-affinity interaction of a biarsenical ligand (FlAsH-EDT2 or ReAsH-EDT2) with the thiols in a tetracysteine (TC) expression tag fused to the protein of interest. The FlAsH-EDT2 ligand is essentially fluorescein that has been modified to contain two arsenic atoms at a set distance from each other, whereas the ReAsH-EDT2 ligand is a similarly modified resorufin (Figure 2.2.11). Virtually nonfluorescent in the ethanedithiol (EDT)-bound state, these reagents become highly fluorescent when bound to the tetracysteine tag Cys-Cys-Xxx-Yyy-Cys-Cys, where Xxx-Yyy is typically Pro-Gly ref (Figure 2.2.12). Modified tags with additional flanking sequences produce higher-affinity binding of the biarsenical ligand, resulting in improved signal-to-background characteristics.ref Selective labeling of two proteins for fluorescence microscopy colocalization and FRET analysis has been accomplished using TC tags with different binding affinities in combination with FlAsH-EDT2 and ReAsH-EDT2.ref Background due to off-target endogenous thiols can be diminished by washing with competitor dithiols such as 2,3-dimercaptopropanol (BAL). Although tetracysteine tag labeling is best suited to reducing intracellular environments, protocols involving co-administration of trialkylphosphine or dithiothreitol (DTT, D1532; Introduction to Thiol Modification and Detection—Section 2.1) reducing agents have been devised for applications in oxidizing environments, including cell surfaces.ref Photosensitized oxidation of diaminobenzidine (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2) by ReAsH enables correlated fluorescence and electron microscopy of tetracysteine-tagged proteins.ref

The six–amino acid tetracysteine tag is less likely to disrupt native protein structure and function than larger tags such as Green Fluorescent Protein ref (GFP, 238 amino acids). Although the majority of TC-FlAsH and TC-ReAsH applications have been in mammalian cells (Figure 2.2.13), the reagents and associated methods are also particularly useful for nondisruptive labeling of viral coat proteins ref and successful adaptations for labeling proteins in yeast,ref bacteria,ref Dictyostelium discoideum ref and plants ref have been described.

Figure 2.2.11
The structures of A) FlAsH-EDT2 ligand and B) ReAsH-EDT2 ligand, which are biarsenical labeling reagents provided in the TC-FlAsH II and TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits (T34561, T34562), respectively.

Figure 2.2.12 Binding of the nonfluorescent FlAsH-EDT2 ligand to a recombinantly expressed tetracysteine sequence yields a highly fluorescent complex.

TC-FlAsH and TC-ReAsH Tetracysteine Tag Detection Kits

Transfecting the host cell line with an expression construct comprising the protein of interest fused to a tetracysteine tag (CCPGCC) is the first step in TC-FlAsH TC-ReAsH detection. The tagged protein is then detected by the addition of FlAsH-EDT2 reagent or ReAsH-EDT2 reagent, which generates green or red fluorescence, respectively, upon binding the tetracysteine motif. For detection of tetracysteine-tagged proteins expressed in cells, we offer the TC-FlAsH II and TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits (T34561, T34562), which provide:

We also offer these TC-FlAsH and TC-ReAsH detection reagents bundled with Gateway expression vectors for use in cloning the tetracysteine-tagged protein fusion. The TC-FlAsH II TC-ReAsH II In-Cell Tetracysteine Tag Detection Kit (with mammalian TC-Tag Gateway expression vectors) (T34563) provides:

In addition to these kits for in-cell detection, we offer the TC-FlAsH Expression Analysis Detection Kits (A10067, A10068; Detecting Protein Modifications—Section 9.4), which are designed for detecting tetracysteine-tagged proteins in polyacrylamide gels (Figure 2.2.14).

Figure 2.2.13
CHO-k1 cells expressing a tetracysteine-tagged version of β-tubulin labeled with FlAsH-EDT2 reagent, provided in the TC-FlAsH II In Cell Tetracysteine Tag Detection Kit (T34561). Upon treatment with vinblastine, a compound known to perturb cytoskeletal structure, tubulin drastically rearranges from A) a reticular structure to B) rod-shaped structures.

Figure 2.2.14 Protein gel staining using TC-FlAsH Expression Analysis Detection Kit (A10068). A) Tetracysteine-tagged proteins are labeled with FlAsH-EDT2 reagent and fluoresce green. B) Total proteins are labeled with the Red total-protein stain provided in the kit and fluoresce red. C) An overlay of the two images reveals relative amounts of protein.

Chromophoric Maleimides and Iodoacetamides

QSY Maleimides and Iodoacetamide

QSY 7 C5-maleimide ref (Q10257, structure) and QSY 9 C5-maleimide (Q30457) are nonfluorescent, thiol-reactive diarylrhodamines with absorption spectra similar to those of our QSY 7 and QSY 9 succinimidyl esters (Q10193, Q20131; Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6; Figure 2.2.15), respectively. Although the QSY 7 and QSY 9 chromophores are spectrally similar, QSY 9 dye exhibits enhanced water solubility. QSY 35 iodoacetamide (Q20348) is a nonfluorescent thiol-reactive analog of the amine-reactive nitrobenzoxadiazole (NBD) dye.

The principal applications of these thiol-reactive QSY derivatives are as nonfluorescent acceptor dyes in fluorescence resonance energy transfer (FRET) assays ref (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). The use of nonfluorescent acceptor dyes avoids the background fluorescence that often results from direct (i.e., nonsensitized) excitation of fluorescent acceptor dyes. The broad and strong absorption of QSY 7 and QSY 9 dyes (absorption maximum ~560 nm, EC ~90,000 cm-1M-1) yields extraordinarily efficient quenching of donors that have blue, green, orange or red fluorescence. QSY 35 derivatives absorb light maximally near 470 nm (Figure 2.2.15), making their conjugates excellent FRET acceptors from UV light–excited donor dyes.

Figure 2.2.15
Normalized absorption spectra of the QSY 35 (blue), QSY 7 (red) and QSY 21 (orange) dyes. The QSY 7 and QSY 9 dyes have essentially identical spectra.


DABMI (D1521, structure) is the thiol-reactive analog of dabcyl succinimidyl ester (D2245, Reagents for Analysis of Low Molecular Weight Amines—Section 1.8) and has similar properties and applications. Its principal application is as a nonfluorescent acceptor dye in fluorescence resonance energy transfer (FRET) assays ref (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). The donor dyes in these assays typically include IAEDANS (I14) and other dyes described in Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3. DABMI is also a useful derivatization reagent for MALDI-MS fragmentation analysis of cysteine-containing peptides.ref

NANOGOLD Monomaleimide

In collaboration with Nanoprobes, Inc. (, we offer thiol-reactive NANOGOLD monomaleimide (N20345) . NANOGOLD particles are small metal cluster complexes of gold particles for research applications in light or electron microscopy.ref These cluster complexes are discrete chemical compounds, not gold colloids. NANOGOLD monomaleimide (N20345) permits attachment of these very small (1.4 nm) yet uniformly sized gold particles to accessible thiol groups in biomolecules (Figure 2.2.16, photo). NANOGOLD monomaleimide, which is supplied as a set of five vials of a powder lyophilized from pH 7.5 HEPES buffer, is simply resuspended with the thiol-containing protein in deionized water at room temperature or below to form the conjugate, after which any excess NANOGOLD monomaleimide is removed by gel filtration.ref

In addition to its many uses for light and electron microscopy, NANOGOLD monomaleimide has been shown to be an extremely efficient quencher for dyes in molecular beacons—probes that can be used for homogeneous fluorescence in situ hybridization assays.ref NANOGOLD 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 microscopy detection.ref

Figure 2.2.16 Reaction of NANOGOLD monomaleimide (N20345) with a thiol. Image courtesy of Nanoprobes, Inc.

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A10254 icon icon 720.66 F,DD,L H2O, DMSO 493 72,000 516 pH 7 1, 2, 3
A10255 icon icon 812.88 F,DD,L H2O, DMSO 528 78,000 552 MeOH 1
A10256 icon icon 908.97 F,DD,L H2O, DMSO 588 96,000 612 pH 7 1, 4
A10258 icon icon 1034.37 F,DD,L H2O, DMSO 554 93,000 570 pH 7 1
A20341 icon icon 880.92 F,DD,L H2O, DMSO 575 92,000 600 pH 7 1, 5
A20342 icon ~1300 F,DD,L H2O, DMSO 622 143,000 640 MeOH 1
A20343 icon ~900 F,DD,L H2O, DMSO 668 112,000 697 MeOH 1, 6
A20344 icon ~1000 F,DD,L H2O, DMSO 684 175,000 714 MeOH 1, 7
A20346 icon ~1250 F,DD,L H2O, DMSO 556 158,000 572 MeOH 1
A20347 icon ~1300 F,DD,L H2O, DMSO 651 265,000 671 MeOH 1, 8
A30459 icon ~1350 F,DD,L H2O, DMSO 753 290,000 783 MeOH 1, 24
B1355 icon 425.23 F,D,L pH >6, DMF 492 81,000 515 pH 9 9
B2103 icon 341.00 F,D,L DMSO, MeCN 533 62,000 561 CHCl3 10, 11
B10250 icon icon 414.22 F,D,L DMSO, MeCN 504 79,000 510 MeOH 11
B10621 icon 840.47 F,D,L DMSO 549 88,000 575 MeOH 12
B20340 icon 788.44 F,D,L DMSO 504 132,000 511 MeOH 13
B22802 icon 449.14 F,D,L DMSO, MeCN 658 73,000 678 CHCl3 14
B30466 icon icon 562.42 F,DD,L DMSO, MeCN 544 60,000 570 MeOH 11
C20260 icon icon 199.55 F,D,L DMF, MeCN 336 9800 none MeOH 15, 16
D1521 icon 320.35 F,D,L DMF, MeCN 419 34,000 none MeOH 17
D2004 icon 419.18 F,D,L DMF, DMSO 478 25,000 541 MeOH 12, 17
D6003 icon icon 417.00 F,D,L DMSO, MeCN 502 76,000 510 MeOH 11, 12
D6004 icon 431.03 F,D,L DMSO, MeCN 508 69,000 543 MeOH 11, 12
D20350 icon 419.24 F,D,L DMSO 499 88,000 508 MeOH 18
D20351 icon 575.38 F,D,L DMSO 577 60,000 618 MeOH 18
E118 icon icon 742.95 F,D,L pH >6, DMF 524 103,000 545 MeOH 1, 19
F150 icon icon 427.37 F,D,L pH >6, DMF 492 83,000 515 pH 9 1, 9, 20
F486 icon 183.10 F,D,L MeCN, CHCl3 328 8000 none MeOH 15
F6053 icon 217.17 F,D,L DMF, DMSO 320 4800 none MeOH 21
I9 icon 406.14 F,D,L DMF, MeCN 472 23,000 536 MeOH 12, 17
I30451 icon icon 515.26 F,D,L pH >6, DMF 492 78,000 515 pH 9 1, 9, 12
I30452 icon icon 515.26 F,D,L pH >6, DMF 491 82,000 516 pH 9 1, 9, 12
L1338 icon icon 659.51 F,D,L H2O 426 11,000 531 pH 7 12
M6026 icon 471.48 F,D,L DMSO 412 23,000 561 MeOH 22
O6010 icon icon 551.24 F,D,L pH >6, DMF 491 68,000 516 pH 9 1, 12, 23
O6034 icon icon 463.35 F,D,L pH >6, DMF 491 81,000 515 pH 9 1, 23
Q10257 icon icon 858.45 F,D,L DMSO 560 92,000 none MeOH  
Q20348 icon icon 453.20 F,D,L DMSO 475 24,000 none MeOH 12
Q30457 icon icon 1083.30 F,D,L H2O, DMSO 562 90,000 none MeOH 1
R6029 icon icon 680.79 F,D,L DMSO 560 119,000 580 MeOH  
T6006 icon icon 825.22 F,D,L DMSO 543 87,000 567 MeOH 12
T6008 icon icon 728.83 F,D,L DMSO 582 112,000 600 MeOH  
T6009 icon icon 811.80 F,D,L DMSO 583 115,000 603 MeOH  
T6027 icon icon 481.51 F,D,L DMSO 541 95,000 567 MeOH  
T6028 icon icon 481.51 F,D,L DMSO 541 91,000 567 MeOH  
T30453 icon 510.31 F,D,L DMSO 503 80,000 510 MeOH  
T30454 icon 658.52 F,D,L DMSO 544 58,000 570 MeOH  
T30455 icon 684.53 F,D,L DMSO 589 63,000 617 MeOH  
T30456 icon 710.57 F,D,L DMSO 625 93,000 640 MeOH  
T34561 icon icon 664.49 FF,D,L,AA DMSO 508 70,000 530 pH 7.2 25, 26
T34562 icon icon 545.37 FF,D,AA DMSO 596 69,000 608 pH 7.2 25, 27
  1. Aqueous stock solutions should be used within 24 hours; long-term storage is NOT recommended.
  2. The fluorescence lifetime (τ) of the Alexa Fluor 488 dye in pH 7.4 buffer at 20°C is 4.1 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.
  3. Abs and Em of the Alexa Fluor 488 dye are red-shifted by as much as 16 nm and 25 nm respectively on microarrays relative to aqueous solution values. The magnitude of the spectral shift depends on the array substrate material.ref
  4. The fluorescence lifetime (τ) of the Alexa Fluor 594 dye in pH 7.4 buffer at 20°C is 3.9 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.
  5. The fluorescence lifetime (τ) of the Alexa Fluor 568 dye in pH 7.4 buffer at 20°C is 3.6 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.
  6. The fluorescence lifetime (τ) of the Alexa Fluor 660 dye in pH 7.5 buffer at 20°C is 1.2 nanoseconds. Data provided by Pierre-Alain Muller, Max Planck Institute for Biophysical Chemistry, Göttingen.
  7. The fluorescence lifetime (τ) of the Alexa Fluor 680 dye in pH 7.5 buffer at 20°C is 1.2 nanoseconds. Data provided by Pierre-Alain Muller, Max Planck Institute for Biophysical Chemistry, Göttingen.
  8. The fluorescence lifetime (τ) of the Alexa Fluor 647 dye in H2O at 20°C is 1.0 nanoseconds and 1.5 nanoseconds in EtOH.ref
  9. Absorption and fluorescence of fluorescein derivatives are pH dependent. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <7.
  10. B2103 spectra are for the unreacted reagent. The thiol adduct has Abs = 493 nm, Em = 503 nm in MeOH.
  11. The absorption and fluorescence spectra of BODIPY derivatives are relatively insensitive to the solvent.
  12. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.
  13. Fluorescence emission of B20340 is relatively weak until the disulfide linkage between its two BODIPY FL fluorophores is reductively cleaved.
  14. B22802 spectral data are for the unreacted reagent. The thiol adduct has Abs = 629 nm, Em = 647 nm in dichloromethane (CH2Cl2).
  15. Spectra of 2-mercaptoethanol adduct of NBD chloride in MeOH: Abs = 425 nm (EC = 13,000 cm-1M-1), Em = 520 nm. NBD fluoride yields the same derivatives as NBD chloride but is more reactive.
  16. This product is specified to equal or exceed 98% analytical purity by HPLC.
  17. Spectral data of the 2-mercaptoethanol adduct.
  18. Spectral data are for the unreacted reagent and are essentially unchanged upon reaction with thiols.
  19. Eosin and erythrosin derivatives also exhibit phosphorescence with an emission maximum at ~680 nm. The phosphorescence lifetime is ~1 millisecond for eosin and 0.5 milliseconds for erythrosin.ref Fluorescence lifetimes (τ) are 1.4 nanoseconds (QY = 0.2) for eosin and 0.1 nanoseconds (QY = 0.02) for erythrosin.ref
  20. QY increases on reaction with thiols; Abs, EC and Em are essentially unchanged.ref
  21. F6053 reaction product with dimethylaminoethanethiol has Abs = 376 nm (EC ~8000 cm-1M-1), Em ~510 nm in MeOH.
  22. Fluorescence emission spectrum shifts to shorter wavelengths in nonpolar solvents.
  23. Absorption and fluorescence of Oregon Green 488 derivatives are pH dependent only in moderately acidic solutions (pH <5).
  24. The fluorescence lifetime (τ) of the Alexa Fluor 750 dye in H2O at 22°C is 0.7 nanoseconds. Data provided by ISS Inc. (Champaign, IL).
  25. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
  26. Data for T34561 represents FlAsH complexed with the tetracysteine peptide FLNCCPGCCMEP.ref The FlAsH-EDT2 reagent is essentially nonfluorescent and has Abs = 496 nm (EC = 69,500 cm-1M-1) in 0.1 M NaOH.ref
  27. Data for T34562 represents ReAsH complexed with the tetracysteine peptide FLNCCPGCCMEP.ref The ReAsH-EDT2 reagent is essentially nonfluorescent and has Abs = 579 nm (EC = 63,000 cm-1M-1) in 0.1 M NaOH.ref
For Research Use Only. Not for use in diagnostic procedures.