The thiol-reactive dyes described in this section have their longest-wavelength absorption peaks at less than 410 nm (Thiol-reactive dyes excited with ultraviolet light—Table 2.2). Typically, these dyes exhibit blue fluorescence and have much weaker absorption than the dyes described in Thiol-Reactive Probes Excited with Visible Light—Section 2.2, with extinction coefficients often below 20,000 cm-1M-1.

The strong environmental dependence of the emission spectra and quantum yields of several of the dyes—especially the coumarin, benzoxadiazole (NBD probes and ABD-F, Thiol-Reactive Probes Excited with Visible Light—Section 2.2), aminonaphthalene (e.g., dansyl) and Dapoxyl fluorophores—makes some of these thiol-reactive probes useful for investigating protein structure and assembly, following protein transport through membranes and studying ligand binding to receptors.ref

Coumarin Derivatives

Alexa Fluor 350 Maleimide

Alexa Fluor 350 C5-maleimide (A30505), a thiol-reactive, sulfonated coumarin derivative (structure), produces protein conjugates that are optimally excited at 346 nm and have bright blue fluorescence emission (spectra) at wavelengths slightly shorter than AMCA or AMCA-X conjugates (spectra) (emission maximum ~442 nm versus 448 nm). The shorter emission maximum of Alexa Fluor 350 conjugates reduces their spectral overlap with the emission of fluorescein and Oregon Green 488 dyes.ref Like our other Alexa Fluor dyes, Alexa Fluor 350 C5-maleimide offers unrivaled brightness and pH-independent fluorescence, as well as water solubility and a low degree of quenching upon conjugation (The Alexa Fluor Dye Series—Note 1.1).

Pacific Blue Maleimide

The Pacific Blue dye, which is based on the 6,8-difluoro-7-hydroxycoumarin fluorophore (structure), exhibits bright blue fluorescence, with excitation/emission maxima of ~410/455 nm. Significantly, the pKa value of this 6,8-difluoro-7-hydroxycoumarin derivative is 2–3 log units lower than that of the corresponding 7-hydroxycoumarin. Thus, the thiol-reactive Pacific Blue C5-maleimide (P30506) yields conjugates that are strongly fluorescent, even at neutral pH. In addition, Pacific Blue conjugates are efficiently excited by the 405 nm spectral line of the violet diode laser developed for fluorescence microscopy and flow cytometry.ref

Other Coumarin Maleimides and Iodoacetamides

We offer several blue-fluorescent thiol-reactive dialkylcoumarins (Thiol-reactive dyes excited with ultraviolet light—Table 2.2), including 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, D346; structure) and N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM, D10251) and the corresponding iodoacetamides DCIA (D404) and DACIA (D10252). The dialkylcoumarin fluorophore is an excellent fluorescence resonance energy acceptor from tryptophan and a good donor to fluorescein, NBD, Alexa Fluor 488 dye, Green Fluorescent Protein (GFP) and the nonfluorescent QSY 7, QSY 9 and QSY 35 quenchers, making these thiol-reactive coumarins especially valuable for studying protein structure and for detecting protein–membrane interactions.ref Fluorescence emission of the dialkylcoumarin conjugates is moderately sensitive to environment.

Unlike MDCC (described below), which is intrinsically fluorescent, the maleimides CPM and DACM are essentially nonfluorescent until they react with thiols, permitting thiol quantitation without a separation step.ref The environment-sensitive fluorescence of CPM is also a useful indicator of protein folding in thermal-shift assays of ligand- and mutagenesis-dependent protein stability ref (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1).

We also offer 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC, D10253; structure). When conjugated to a mutant phosphate-binding protein, MDCC has proven useful for direct, real-time measurement of inorganic phosphate release during enzymatic reactions.ref Similarly, environment-sensitive fluorescence of MDCC site-specifically attached to Escherichia coli ParM nucleotide-binding protein provides the basis for a fluorogenic ADP biosensor.ref

Pacific Orange Maleimide

The thiol-reactive Pacific Orange C5-maleimide (P30507) yields conjugates with excitation/emission maxima of ~400/551 nm, making it ideal for use with violet diode laser–equipped flow cytometers and fluorescence microscopes. Moreover, Pacific Blue conjugates (described above with the other coumarins) and Pacific Orange conjugates can be simultaneously excited at 405 nm and emit at 455 and 551 nm, respectively, facilitating two-color analysis.ref

Pyrene Derivatives

Pyrene Maleimide

Not only is N-(1-pyrene)maleimide (pyrene maleimide, P28; structure) essentially nonfluorescent until it has reacted with thiols, but once excited, pyrene–thiol conjugates can interact to form excited-state dimers (excimers) that emit at longer wavelengths than the excited monomeric fluorophore. Pyrene maleimide conjugates often have very long fluorescence lifetimes (>100 nanoseconds), giving proximal pyrene rings within 6–10 Å of each other ample time to form the spectrally altered excimer (Figure 2.3.1). The excimer-forming capacity of pyrene maleimide can be exploited for detection of conformational changes ref and subunit assembly ref of proteins and for analysis of protease activity.ref

Figure 2.3.1
Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen.

Pyrene Iodoacetamides

Fluorescence of the actin monomer labeled with pyrene iodoacetamide (P29, structure) has been demonstrated to change upon polymerization, making this probe a widely utilized tool for following the kinetics of actin polymerization.ref Conjugates of N-(1-pyrenemethyl)iodoacetamide (P2007MP, structure) have the longest excited-state fluorescence lifetimes (>100 nanoseconds) of all reported thiol-reactive probes. Excimer formation can also be a useful indicator of protein folding.ref

Naphthalene Derivatives

Acrylodan and Badan

As compared with iodoacetamides or maleimides, acrylodan (A433, structure) and 6-bromoacetyl-2-dimethylaminonaphthalene (badan, B6057, structure) generally react with thiols more slowly but they form very strong thioether bonds that are expected to remain stable under conditions required for complete amino acid analysis. The fluorescence emission peak and intensity of these adducts (Figure 2.3.2) are particularly sensitive to conformational changes or ligand binding, making these dyes some of the most useful thiol-reactive probes for protein structure studies.ref For example, the acrylodan conjugate of an intestinal fatty acid–binding protein, ADIFAB (A3880, Probes for Lipid Metabolism and Signaling—Section 17.4), is a sensor for free fatty acids ref (Figure 2.3.3).

Figure 2.3.2 Fluorescence emission spectra of the 2-mercaptoethanol adduct of badan (B6057) in: 1) toluene, 2) chloroform, 3) acetonitrile, 4) ethanol, 5) methanol and 6) water. Each solution contains the same concentration of the adduct. Excitation of all samples is at 380 nm.

Figure 2.3.3 Ribbon representation of the ADIFAB free fatty acid indicator (A3880). In the left-hand image, the fatty acid binding site of intestinal fatty acid–binding protein (yellow) is occupied by a covalently attached acrylodan fluorophore (blue). In the right-hand image, a fatty acid molecule (gray) binds to the protein, displacing the fluorophore (green) and producing a shift of its fluorescence emission spectrum. Image contributed by Alan Kleinfeld, FFA Sciences LLC, San Diego.


To develop appreciable fluorescence, both the reactive anilinonaphthalenesulfonate iodoacetamide (IAANS, I7; structure) and maleimide (MIANS, also called Mal-ANS; M8, structure) must be reacted with thiols that are located in hydrophobic sites. Often, however, buried unsolvated thiol residues are exceptionally reactive, allowing these sites to be selectively modified by these reagents. The environment-sensitive fluorescence properties of the protein conjugates of MIANS and IAANS are similar to those of the structurally related probes 1,8-ANS and 2,6-TNS (A47, T53; Other Nonpolar and Amphiphilic Probes—Section 13.5). The fluorescence intensity, and to a lesser extent, the emission wavelengths of the conjugates, tend to be very sensitive to substrate binding and folding and unfolding of the protein, as well as the association of the labeled protein with other proteins, membranes or nucleic acids.ref


The fluorescence of IAEDANS (I14, structure) is quite dependent upon environment,ref although less so than that of IAANS and MIANS conjugates. Its conjugates frequently respond to ligand binding by undergoing spectral shifts and changes in fluorescence intensity that are determined by the degree of aqueous solvation. Advantages of this reagent include high water solubility above pH 4 and a relatively long fluorescence lifetime (sometimes >20 nanoseconds, although commonly 10–15 nanoseconds), making the conjugates useful for fluorescence polarization assays ref (Fluorescence Polarization (FP)—Note 1.4). The emission spectrum of IAEDANS overlaps well with the absorption of fluorescein, Alexa Fluor 488 and Oregon Green 488 dyes, as well as that of Green Fluorescent Protein (GFP). IAEDANS is an excellent reagent for fluorescence resonance energy transfer (FRET) measurements ref (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). IAEDANS usually reacts with thiols; however, it has been reported to react with a lysine residue in tropomyosin.ref

Bimanes for Thiol Derivatization

Monobromobimane and Monochlorobimane

Monobromobimane (M1378, M20381; structure), which is essentially nonfluorescent until conjugated, readily reacts with low molecular weight thiols,ref including glutathione.ref This reagent, originally described by Kosower and colleagues,ref is also useful for detecting the distribution of protein thiols in cells before and after chemical reduction of disulfides.ref Monobromobimane is reportedly susceptible to inactivation by the disulfide reducing agent TCEP ref (T2556, Introduction to Thiol Modification and Detection—Section 2.1). Both monobromobimane and the more thiol-selective monochlorobimane (M1381MP) have been extensively used for detecting glutathione in live cells ref (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6).


Dibromobimane (D1379, structure) is an interesting homobifunctional crosslinking reagent for proteins ref because it is unlikely to fluoresce until both of its alkylating groups have reacted. It has been used to crosslink thiols in myosin,ref Escherichia coli lactose permease ref and P-glycoprotein.ref Despite its short length, dibromobimane is also an effective intermolecular crosslinker in some cases.ref

Bimane Iodoacetamide and Maleimide

Bimane iodoacetamide (B30500, structure) and bimane C3-maleimide (B30501, structure) are blue-fluorescent thiol-reactive fluorophores with excitation/emission maxima of ~375/456 nm. The small size of the bimane fluorophore reduces the likelihood that the label will interfere with the function of the biomolecule, an important advantage for site-selective probes.

Polar Reagents for Determining Thiol Accessibility

Like IAEDANS (I14), the iodoacetamide and maleimide derivatives of stilbene (A484, A485) have high water solubility and are readily conjugated to thiols. Their combination of high polarity and membrane impermeability makes these polysulfonated dyes useful for determining whether thiol-containing proteins and polypeptide chains are exposed at the extracellular or cytoplasmic membrane surface.

The sulfonated stilbene iodoacetamide (A484, structure) was used to label single-cysteine mutants of staphylococcal α-hemolysin in order to determine structural changes that occur during oligomerization and pore formation ref and of the lipid-binding region of E. coli pyruvate oxidase in order to detect conformational changes upon substrate binding.ref Similarly, single-cysteine mutants of Escherichia coli Na+-glutamate transporter GHS have been probed with the sulfonated stilbene maleimide (A485, structure) to systematically study the topology of this membrane protein.ref

1,10-Phenanthroline Iodoacetamide for Preparing Metal-Binding Conjugates

Conjugation of N-(1,10-phenanthrolin-5-yl)iodoacetamide (P6879, structure) to thiol-containing ligands confers the metal-binding properties of this important complexing agent on the ligand. For example, the covalent copper–phenanthroline complex of oligonucleotides or nucleic acid–binding molecules in combination with hydrogen peroxide acts as a chemical nuclease to selectively cleave DNA or RNA.ref

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A433 icon 225.29 L DMF, MeCN 391 20,000 500 MeOH 1
A484 icon 624.33 F,D,L H2O 329 39,000 408 pH 8 2, 3
A485 icon 536.44 F,D H2O 322 35,000 411 pH 8 2
A30505 icon icon 578.68 F,DD,L H2O, DMSO 345 17,000 444 pH 7 4
B6057 icon 292.17 F,L DMF, MeCN 387 21,000 520 MeOH 5
B30500 icon 375.17 F,D,L DMSO 375 5800 456 MeOH 3
B30501 icon 358.35 F,D,L DMSO 375 5700 458 MeOH  
D346 icon 402.45 F,D,L DMSO 384 33,000 469 MeOH 6
D404 icon 490.34 F,D,L DMSO 384 31,000 470 MeOH 2, 3
D1379 icon icon 350.01 L DMF, MeCN 391 6100 see Notes MeOH 7
D10251 icon 298.30 F,D,L DMSO 383 27,000 463 MeOH 8
D10252 icon 386.19 F,D,L DMSO 376 24,000 465 MeOH 3
D10253 icon 383.40 F,D,L DMSO 419 50,000 466 MeOH 9
I7 icon 504.27 F,D,L DMF 326 27,000 462 MeOH 2, 3
I14 icon 434.25 F,D,L pH >6, DMF 336 5700 490 pH 8 3, 10
M8 icon 416.38 F,D,L DMSO, DMF 322 27,000 417 MeOH 11
M1378 icon icon 271.11 F,L DMF, MeCN 398 5000 see Notes pH 7 7
M1381MP icon icon 226.66 F,L DMSO 380 6000 see Notes MeOH 7
M20381 icon icon 271.11 F,L DMF, MeCN 398 5000 see Notes pH 7 7, 12
P28 icon 297.31 F,D,L DMF, DMSO 338 40,000 375 MeOH 13, 14
P29 icon 385.20 F,D,L DMF, DMSO 339 26,000 384 MeOH 2, 3
P2007MP icon 399.23 F,D,L DMSO 341 41,000 377 MeOH 2, 3, 14
P6879 icon 363.16 F,D,L DMSO 270 28,000 none CHCl3 3
P30506 icon icon 406.34 F,DD,L DMSO 402 40,000 451 pH 9 15
P30507   ~800 F,DD,L DMSO 403 23,000 552 MeOH  
  1. Fluorescence of unconjugated A433 is weak, increasing markedly upon reaction with thiols. Em (QY) for the 2-mercaptoethanol adduct are: 540 nm (0.18) in H2O, 513 nm (0.57) in MeOH, 502 nm (0.79) in EtOH, 468 nm (0.78) in MeCN, 435 nm (0.83) in dioxane.ref
  2. Spectral data of the 2-mercaptoethanol adduct.
  3. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.
  4. Aqueous stock solutions should be used within 24 hours; long-term storage is NOT recommended.
  5. Em for 2-mercaptoethanol adduct of B6057: 550 nm in H2O (pH 7), 523 nm in MeOH, 514 nm in EtOH, 502 nm in MeCN, 469 nm in CHCl3, 457 nm in dioxane, 445 nm in toluene. Abs is relatively independent of solvent.
  6. Spectral data are for the 2-mercaptoethanol adduct. The unreacted reagent is nonfluorescent, Abs = 384 nm (EC = 32,000 cm-1M-1) in MeOH.
  7. Bimanes are almost nonfluorescent until reacted with thiols. For monobromobimane conjugated to glutathione, Abs = 394 nm, Em = 490 nm (QY ~0.1–0.3) in pH 8 buffer.ref
  8. Spectral data are for the 2-mercaptoethanol adduct. The unreacted reagent is nonfluorescent, Abs = 381 nm (EC = 27,000 cm-1M-1) in MeOH.
  9. QY increases on reaction with thiols; Abs, EC and Em are unchanged.ref
  10. The 2-mercaptoethanol adduct of I14 has essentially similar spectral characteristics in aqueous solution.ref Fluorescence lifetime (τ) = 21 nsec when conjugated to myosin subfragment-1.ref
  11. Spectral data are for the 2-mercaptoethanol adduct. The unreacted reagent is nonfluorescent, Abs = 443 nm (EC = 13,000 cm-1M-1) in MeOH.
  12. This product is specified to equal or exceed 98% analytical purity by HPLC.
  13. Fluorescence of unreacted P28 is weak. Em data represent the 2-mercaptoethanol adduct.
  14. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below 300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations.
  15. The fluorescence quantum yield of Pacific Blue dye in 50 mM potassium phosphate, 150 mM NaCl, pH 7.2, at 22°C is 0.78.