Shop All Thiol-Reactive Fluorophores, Biotins & Other Labels

IANBD Amide (N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine) (Invitrogen™)

The thiol-reactive IANBD amide can be used to create environment sensitive probes. The absorption and fluorescence emission spectra, quantum yields, and extinction coefficients of NBD conjugates are all markedly dependent on solvent. Additionally, NBD is also a function analog of the dinitrophenyl hapten, and its fluorescence is quenched upon binding to anti-dinitrophenyl (anti-DNP) antibodies.

BODIPY™ TMR C5-Maleimide (Invitrogen™)

The thiol-reactive BODIPY® TMR maleimide produces electronically neutral dye conjugates that are spectrally similar to the positively charged tetramethylrhodamine dye. This dye's lack of ionic charge results in minimal effects on the isoelectric points of standard proteins conjugated with this fluorophore. The small size and relatively long excited-state lifetime of BODIPY® TMR dye has proven useful for studying ligand-receptor interaction by fluoresence polarization.

DACM, N-(7-Dimethylamino-4-Methylcoumarin-3-yl))Maleimide (Invitrogen™)

The thiol reactive coumarin, DACM can be used to create blue-fluorescent bioconjugates. When compared with AMCA conjugates, conjugates of the UV light-excitable 7-dialkylaminocoumarin fluorophore have slightly longer-wavelength emission spectra (~470 nm). The unreacted reagent is nonfluorescent and can also be used to quantitate free thiols.

Acrylodan (6-Acryloyl-2-Dimethylaminonaphthalene) (Invitrogen™)

The thiol reactive acrylodan (6-acryloyl-2-dimethylaminonaphthalene) 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 acrylodan conjugates may make this useful for distinguishing thiols that are located in membranes versus those exposed to aqueous solvation in cells.

Alexa Fluor™ 488 C5 Maleimide (Invitrogen™)

Alexa Fluor® 488 is a bright, green fluorescent dye with excitation ideally suited to the 488 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 488 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 488 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® 488 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® 488 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® 488 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 493/516 nm
Extinction coefficient: 72,000 cm-1M-1
Spectrally similar dyes: Fluorescein (FITC), Cy®2
Molecular weight: 720.66

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.

MDCC, 7-Diethylamino-3-((((2-Maleimidyl)ethyl)amino)carbonyl)coumarin (Invitrogen™)

The thiol reactive coumarin, MDCC can be used to create blue-fluorescent bioconjugates. When compared with AMCA conjugates, conjugates of the UV light-excitable 7-dialkylaminocoumarin fluorophore have slightly longer-wavelength emission spectra (~470 nm).

Fluorescein-5-Maleimide (Invitrogen™)

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

EZ-Link™ Iodoacetyl-PEG2-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Iodoacetyl-PEG2-Biotin is a mid-length, haloacetyl-activated, sulfhydryl-reactive biotinylation reagent that contains a 2-unit ethylene glycol in its spacer arm for increased water-solubility characteristics.

Features of EZ-Link Iodoacetyl-PEG2-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
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
Pegylated—spacer arm contains a hydrophilic, 2-unit, polyethylene glycol (PEG) group
Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—can be dissolved directly in aqueous buffers for labeling reactions
Medium length—spacer arm (total length added to target) is 24.7 angstroms

Iodoacetyl-PEG2-Biotin enables simple and efficient biotin labeling of antibodies, cysteine-containing peptides and other thiol-containing molecules. The iodoacetyl group reacts with reduced thiols (sulfhydryl groups,—SH) at alkaline pH to form stable thioether bond. The hydrophilic, 2-unit polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled proteins stored in solution. The PEG segment adds length and flexibility to the spacer arm, minimizing steric hindrance involved with binding to avidin molecules.

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.

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

Thermo Scientific EZ-Link HPDP-Biotin is pyridyldithiol-activated, sulfhydryl-reactive biotinylation reagent that conjugates via a cleavable (reversible) disulfide bond to enable use in a variety of purification methods.

Features of EZ-Link HPDP-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
Thiol-reactive—reacts with sulfhydryls (-SH), such as the side-chain of cysteine (C)
Pyridyldithiol-activated—perform reactions at pH 6.5 to 7.5 in buffers such as PBS
Reversible—forms disulfide bonds, which can be cleaved using DTT or other reducing agent
Solubility—must be dissolved in DMSO or DMF before further dilution in aqueous buffers
Medium length—spacer arm (total length added to target) is 29.2 angstroms

HPDP-Biotin is a pyridyldithiol-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 reversible disulfide bonds. HPDP-Biotin is useful for labeling and affinity-purification applications that require recovery of the original, unmodified molecule. For example, a protein can be biotinylated, allowed to bind its interactor, then captured to a streptavidin column and finally eluted and recovered by reduction of the disulfide bond with dithiothreitol.

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. Pyridyldithiol reagents specifically react with sulfhydryl groups (-SH) in near-neutral buffers to form reversible disulfide 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:
• Retrieve cell surface receptors and cleave the biotin away on an immobilized avidin column

SAMSA Fluorescein, 5-((2-(and-3)-S-(acetylmercapto) succinoyl) amino) Fluorescein, mixed isomers (Invitrogen™)

SAMSA fluorescein is a useful reagent for forming fluorescent protein conjugates and for assaying maleimide and iodoacetamide moieties on proteins with fluorescein. SAMSA fluorescein is activated with base to remove the acetyl protecting group, thereby generating a thiol-containing fluorescein.

Alexa Fluor™ 532 C5 Maleimide (Invitrogen™)

Alexa Fluor® 532 is a bright, yellow fluorescent dye with excitation ideally suited for the frequency-doubled Nd:YAG laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 532 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 532 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® 532 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® 532 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® 532 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 528/552 nm
Extinction coefficient: 78,000 cm-1M-1
Molecular weight: 812.88

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.

DyLight™ 680 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 680 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 680 used to fluorescently label sulfhydryl-containing peptides, proteins and other biomolecular probes.

DyLight 680 produces near-infrared (IR) fluorescence that replaces other near-IR dyes, including Cy5.5™ dye and Alexa Fluor™ 680, and is ideal for multi-color 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 680 Maleimide:

High performance—DyLight 680 fluoresces brighter than Alexa Fluor 680 and Cy5.5 dyes
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 680 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.

pHrodo™ Green Maleimide (Invitrogen™)

The thiol-reactive pH-sensitive pHrodo® Green Maleimide dye is suitable for the creation of bioconjugates to study endocytosis and phagocytosis. pHrodo® Green dramatically increases fluorescence as the pH of its surroundings become more acidic.
• Use pH-sensitive pHrodo® Green Maleimide to make pH-sensitive bioconjugates of your choice
• Get faster, more accurate results than with any other endocytosis or phagocytosis assay—no need for wash steps or quenchers
• Multiplex with red fluorescent markers such as RFP, pHrodo® Red, and many others

The increase in fluorescence of pHrodo® Green as pH changes from basic to acidic correlates with the acidification of intracellular vesicles, making it an ideal tool to study endocytosis or phagocytosis and their regulation by environmental factors, drugs, or pathogens. The spectral properties of pHrodo® Green makes it useful for multi-color experiments. pHrodo® Green can be detected using most standard green fluorescent filter sets and has been validated for use on a variety of platforms including flow cytometry, fluorescent microscopy, and high content screening (HCS). The lack of fluorescence of pHrodo® Green in a typical extracellular environment eliminates the need for wash steps or quencher dyes in the experimental workflow.

pHrodo® Green Maleimide is a thiol-reactive dye that can be used to create pHrodo® Green bioconjugates in aqueous buffer. The maleimide reacts with free sulphydryl groups produced by the reduction of cysteines in proteins or peptides. Maleimides are particularly useful for labeling antibodies as the dye will not attach to the antibody binding site. This reaction will result in a stable conjugate that can be used in live cell assays or stored for later use.

pHrodo® Green is also available in an amine-reactive form (see pHrodo® Green STP), as well as a selection of ready-to-use conjugates (e.g., E. coli, S. aureus, and dextran). In addition, pHrodo® Red reactive dyes and ready-to-use conjugates are available as a color alternative with the same properties.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

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

Tetramethylrhodamine-5-Maleimide, single isomer (Invitrogen™)

Tetramethylrhodamine-5-maleimide is a thiol-reactive dye that yields photostable, pH-insensitive, red-orange—fluorescent conjugates.