Shop All Thiol-Reactive Fluorophores, Biotins & Other Labels

DyLight™ 550 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 550 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 550 used to fluorescently label sulfhydryl-containing peptides, proteins and other biomolecular probes.

DyLight 550 provides vibrant orange-to-red fluorescence with better performance than other rhodamine derivatives including Alexa Fluor™ 555, TRITC and Cy3™ dye for fluorescent applications over a broad pH range (pH 4-9). The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates.

Features of DyLight 550 Maleimide:

High performance—DyLight 550 shows brighter fluorescence than Alexa Fluor 555, TRITC and Cy3 dye
Specific—maleimide-activated dye labels proteins and other molecules at reduced sulfhydryls (-SH)
Efficient labeling methods—well-characterized chemistry and optimized protocols provide for reliable, high-quality labeling
Optimized antibody labeling procedure—complete protocol for IgG reduction and labeling and calculating the labeling efficiency

Applications:
• Antibody labeling for immunofluorescence applications, including immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting and ELISA assay
• Target macromolecule labeling for in vitro and in vivo fluorescent detection strategies

DyLight 550 Sulfhydryl-Reactive Dye is activated with a maleic acid imide (maleimide) moiety to form a reactive alkylation reagent. Labeling occurs through reaction of the maleimide-activated dye with reduced sulfhydryl groups (-SH) to form stable thioether bonds. Maleimides are specific for sulfhydryl groups between pH 6.5-7.5. Learn more about maleimide chemistry.

Fluorescein-5-Maleimide (Invitrogen™)

Searching for superior alternatives to fluorescein? Our Alexa Fluor Dye Series offers everything you're looking for and more.

DTNB; Ellman's Reagent, 5,5'-Dithiobis-(2-Nitrobenzoic Acid) (Invitrogen™)

DTNB or Elman's reagent can be used to quantitate thiols in proteins, cells and plasma by absorption measurements. It readily forms a mixed disulfide with thiols, liberating the chromophore 5-merapto-2-nitrobenzoic acid (absorption maximum 410 nm). Only protein thiols that are accessible to this water-soluble reagent are modified.

DyLight™ 350 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 350 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 350 used to fluorescently label sulfhydryl-containing peptides, proteins and other biomolecular probes.

DyLight 350 has high fluorescence intensity over a broad pH range (pH 4-9) and is more photostable than Alexa Fluor™ 350 and AMCA dyes in many applications. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates.

Features of DyLight 350 Sulfhydryl-Reactive Dye:

High performance—DyLight 350 shows brighter fluorescence than Alexa Fluor 350 and AMCA
Specific—maleimide-activated dye labels proteins and other molecules at reduced sulfhydryls (-SH)
Efficient labeling methods—well-characterized chemistry and optimized protocols provide for reliable, high-quality labeling
Optimized antibody labeling procedure—complete protocol for IgG reduction and labeling and calculating the labeling efficiency

Applications:
• Antibody labeling for immunofluorescence applications, including immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting and ELISA assay
• Target macromolecule labeling for in vitro and in vivo fluorescent detection strategies

DyLight 350 Sulfhydryl-Reactive Dye is activated with a maleic acid imide (maleimide) moiety to form a reactive alkylation reagent. Labeling occurs through reaction of the maleimide-activated dye with reduced sulfhydryl groups (-SH) to form stable thioether bonds. Maleimides are specific for sulfhydryl groups between pH 6.5-7.5. Learn more about maleimide chemistry.

Related Products
DyLight™ 350 Maleimide

Thiol and Sulfide Quantitation Kit (Invitrogen™)

The Thiol and Sulfide Quantitation Kit provides an ultrasensitive colorimetric assay for quantitating both protein and nonprotein thiols (also called sulfhydryls or mercaptans). In this assay, thiols or inorganic sulfides reduce a disulfide-inhibited derivative of papain, stoichiometrically releasing the active enzyme. The activity of the enzyme is then measured using the chromogenic papain substrate N-benzoyl-L-argining, p-nitroanilide (L-BAPNA). The enzymatic amplification step in this kit enables researchers to detect as little as 0.2 µM thiol - a sensitivity that is about 100-fold greater than that achieved using Ellman's reagent.

EZ-Link™ BMCC-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link BMCC-Biotin is a maleimide-activated, sulfhydryl-reactive biotinylation reagent with an extended spacer arm that contains a stabilizing cyclohexane group.

Features of EZ-Link BMCC-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
Membrane-permeable—can be used to label inside cells (intracellular)
Thiol-reactive—reacts with sulfhydryls (-SH), such as the side-chain of cysteine (C)
Maleimide-activated—perform reactions at pH 6.5 to 7.5 in buffers such as PBS
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—must be dissolved in DMSO or DMF before further dilution in aqueous buffers
Medium length—spacer arm (total length added to target) is 32.6 angstroms; contains cyclohexane ring, which stabilizes adjacent maleimide

BMCC-Biotin is a maleimido-biotin compound for labeling protein cysteines and other molecules that contain sulfhydryl groups. This reagent specifically reacts with reduced thiols (-SH) in near-neutral buffers to form permanent (irreversible) thioether bonds. The unique feature of BMCC-Biotin is its spacer arm cyclohexane ring; this has a stabilizing effect that minimizes hydrolysis and degradation of the maleimide group until it has opportunity to conjugate with target thiols.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

Biotinylation reagents differ in reactivity, length, solubility, cell permeability and cleavability. Three types of sulfhydryl-reactive compounds are available: maleimido, iodoacetyl and pyridyldithiol. Maleimide reagents specifically react with sulfhydryl groups (-SH) in near-neutral buffers to form permanent thioether bonds.

In proteins, sulfhydryls exist where there are cysteine (C) residues. Cystine disulfide bonds must be reduced to make sulfhydryl groups available for labeling. Hinge-region disulfide bridges of antibodies can be selectively reduced to make functional half-antibodies that can be labeled.

EZ-Link™ Iodoacetyl-LC-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Iodoacetyl-LC-Biotin is a mid-length, haloacetyl-activated, sulfhydryl-reactive biotinylation reagent that forms stable, irreversible thioether bonds at alkaline pH.

Features of EZ-Link Iodoacetyl-LC-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
Membrane-permeable—can be used to label inside cells (intracellular)
Thiol-reactive—reacts with sulfhydryls (-SH), such as the side-chain of cysteine (C)
Iodoacetyl-activated—perform reactions in the dark at pH 7.5 to 8.5 in Tris or borate buffer
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—must be dissolved in DMSO or DMF before further dilution in aqueous buffers
Medium length—spacer arm (total length added to target) is 27.1 angstroms; contains hexylenediamine extension

Iodoacetyl-LC-Biotin is a haloacetyl-biotin compound for labeling protein cysteines and other molecules that contain sulfhydryl groups. This reagent specifically reacts with reduced thiols (-SH) in alkaline buffers to form permanent (irreversible) thioether bonds. The unique feature of Iodoacetyl-LC-Biotin is its extended yet chemically simple hexylenediamine spacer arm.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

Biotinylation reagents differ in reactivity, length, solubility, cell permeability and cleavability. Three types of sulfhydryl-reactive compounds are available: maleimido, iodoacetyl and pyridyldithiol. Iodoacetyl reagents specifically react with sulfhydryl groups (-SH) at pH 8.3 to form permanent thioether bonds.

In proteins, sulfhydryls exist where there are cysteine (C) residues. Cystine disulfide bonds must be reduced to make sulfhydryl groups available for labeling. Hinge-region disulfide bridges of antibodies can be selectively reduced to make functional half-antibodies that can be labeled.

Applications:
• Electron microscopy studies on spatial relationships between proteins (Ref.1)
• Localizing the SH1 thiol of the myosin head using avidin-biotin complexes in electron microscopy (Ref.2)

Monobromobimane (mBBr), 25 mg (Invitrogen™)

Monobromobimane is essentially nonfluorescent until conjugated, readily reacts with several low molecular weight thiols, including glutathione, N-acetylcysteine, mercaptopurine, peptides and plasma thiols. The glutathione conjugate of monobromobimane has absorption/emission maxima ~394/490 nm.

Badan (6-Bromoacetyl-2-Dimethylaminonaphthalene) (Invitrogen™)

The thiol reactive badan generally reacts with thiols more slowly than iodoacetamides or maleimides, but does 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 are particularly sensitive to conformational changes or ligand binding, making this dye one of the most useful thiol-reactive probe for protein structure studies. The environment-sensitive spectral shift of badan conjugates may make this useful for distinguishing thiols that are located in membranes versus those exposed to aqueous solvation in cells.

Alexa Fluor™ 350 C5 Maleimide (Invitrogen™)

Alexa Fluor® 350 is a blue fluorescent dye with moderate photostability and excitation that matches the 350 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 350 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 350 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The maleimide derivative of Alexa Fluor® 350 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor® 350 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® maleimide:

Fluorophore label: Alexa Fluor® 350 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 345/444 nm
Extinction coefficient: 17,000 cm-1M-1
Spectrally similar dyes: Marina Blue
Molecular weight: 578.68

Typical Conjugation Reaction
The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation.

The Alexa Fluor® maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Alexa Fluor™ 555 C2 Maleimide (Invitrogen™)

Alexa Fluor® 555 is a bright orange dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 555 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 555 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The maleimide derivative of Alexa Fluor® 555 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor® 555 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® maleimide:

Fluorophore label: Alexa Fluor® 555 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 556/572 nm
Extinction coefficient: 158,000 cm-1M-1
Spectrally similar dyes: Tetramethylrhodamine
Molecular weight: ~1250

Typical Conjugation Reaction
The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation.

The Alexa Fluor® maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

N-(1-Pyrene)Maleimide (Invitrogen™)

The thiol-reactive N-(1-pyrene)maleimide can be used to create environment-sensitive bioconjugates with this unique fluorophore.

DyLight™ 755-4xPEG Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 755-4xPEG Sulfhydryl-Reactive Dye is a maleimide-activated derivative of our high-performance DyLight 755 Dye used to fluorescently label cysteine-containing peptides, proteins or other biomolecular probes.

The DyLight 755-4xPEG dye contains 4 polyethylene glycol (PEG) chains that are non-cytotoxic, enhance fluorescence and reduce nonspecific binding of conjugates made with them. Conjugates made with DyLight 755-4xPEG Dye can be used as molecular probes for cellular imaging and other fluorescence detection methods. The NIR fluorescence properties of DyLight 755-4xPEG Dye make it especially useful in a variety of biological, chemical, and pharmaceutical applications, including in vivo imaging. The PEG chains also improve solubility of the dyes and labeled molecules in aqueous solution, aid in cell permeability and improve tissue retention.

Features of DyLight 755-4xPEG Maleimide:

High fluorescence intensity—significantly brighter fluorescence than Alexa Fluor™ 750
PEGylated—improves solubility in aqueous solution and aids in cell permeability
Specific—maleimide-activated dye labels proteins and other molecules at reduced sulfhydryls (-SH)

Applications:
• Fluorescence microscopy
In vivo or ex vivo imaging
• Cell-based assays
• Flow cytometry/fluorescence-activated cell sorting (FACS)

DyLight 755-4xPEG Sulfhydryl-Reactive Dye is activated with a maleic acid imide (maleimide) moiety to form a reactive alkylation reagent. Labeling occurs through reaction of the maleimide-activated dye with reduced sulfhydryl groups (-SH) to form stable thioether bonds. Maleimides are specific for sulfhydryl groups between pH 6.5-7.5. Learn more about maleimide chemistry.

IAEDANS (1,5-IAEDANS, 5-((((2-Iodoacetyl)amino)ethyl)amino)Naphthalene-1-Sulfonic Acid) (Invitrogen™)

The fluorescence of the thiol-reactive IAEDANS is quite dependent upon environment. 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 (10-15 nanoseconds), making the conjugates useful for fluorescence polarization and rotational studies. In addition, because it has a large Stokes shift and an emission that overlaps with the absorption of fluorescein, Alexa Fluor® 488, Oregon Green® dyes and BODIPY® FL dyes, IAEDANS is an excellent reagent for fluorescence resonance energy transfer (FRET) measurements.

5-IAF (5-Iodoacetamidofluorescein) (Invitrogen™)

The thiol-reactive 5-iodoacetamidofluorescein (5-IAF) can be used to produce bioconjugates with the 5-isomer of fluorescein.