Shop All Thiol-Reactive Fluorophores, Biotins & Other Labels

N-(1-Pyrene)Iodoacetamide (Invitrogen™)

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

DyLight™ 800-4xPEG Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 800-4xPEG Sulfhydryl-Reactive Dye is a maleimide-activated derivative of our high-performance DyLight 800 Dye used to fluorescently label cysteine-containing peptides, proteins or other biomolecular probes.

The DyLight 800-4xPEG 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 800-4xPEG Dye can be used as molecular probes for cellular imaging and other fluorescence detection methods. The NIR fluorescence properties of DyLight 800-4xPEG 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 800-4xPEG Maleimide:

High fluorescence intensity—fluorescence comparable to Alexa Fluor™ 800 and IRDye™ 800
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 800-4xPEG 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.

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™ 755 Maleimide (Thermo Scientific™)

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

DyLight 755 is a near-IR fluor that is invisible to the naked eye but increases the staining options when using infrared imaging systems. DyLight 755 has spectral properties that are very similar to other near-IR dyes, including Alexa Fluor™ 750. 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 755 Maleimide:

High performance—DyLight 755 replaces Alexa Fluor 755 for near-infrared staining
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 755 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.

DyLight™ 633 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 633 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 633 used to fluorescently label sulfhydryl-containing peptides, proteins, and other biomolecular probes.

DyLight 633 fluoresces red and has physical properties comparable to other 633 dyes, including Alexa Fluor™ 633, 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 633 Maleimide:

High performance—DyLight 633 shows brighter fluorescence than Alexa Fluor 633
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 633 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.

Alexa Fluor™ 594 C5 Maleimide (Invitrogen™)

Alexa Fluor® 594 is a bright, red fluorescent dye that can be excited using the 561 nm or 594 nm laser lines. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 594 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 594 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® 594 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® 594 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® 594 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 588/612 nm
Extinction coefficient: 96,000 cm-1M-1
Spectrally similar dyes: Texas Red
Molecular weight: 908.97

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.

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.

Tetramethylrhodamine-5-Iodoacetamide Dihydroiodide (5-TMRIA), single isomer (Invitrogen™)

The thiol-reactive tetramethylrhodamine-5-iodoacetamide dihydroiodide (5-TMRIA) can be used to can be used to create bright orange-red-fluorescent bioconjugates with excitation/emission maxima ~555/580.

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.

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).

4-Acetamido-4'-((iodoacetyl)amino)Stilbene-2,2'-Disulfonic Acid, Disodium Salt (Invitrogen™)

The iodoacetamide derivative of stilbene (4-acetamido-4'-((iodoacetyl) amino)stilbene-2,2'-disulfonic acid, disodium salt) is readily conjugated to thiols. The combination of high polarity and membrane impermeability makes this polysulfonated dye useful for determining whether thiol-containing proteins and polypeptide chains are exposed at the extracellular or cytoplsamic membrane surface. Stilbene protein adducts are charged and can be detected by gel or capillary electrophoresis.

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™ 546 C5 Maleimide (Invitrogen™)

Alexa Fluor® 546 is a bright, orange fluorescent dye that can be excited using the 488 nm or 532 nm laser lines. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 546 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 546 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® 546 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® 546 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® 546 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 554/570 nm
Extinction coefficient: 93,000 cm-1M-1
Spectrally similar dyes: Rhodamine red, Cy3
Molecular weight: 1034.37

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.

BODIPY™ FL Maleimide (BODIPY™ FL N-(2-Aminoethyl))Maleimide) (Invitrogen™)

The thiol-reactive BODIPY® FL maleimide produces electronically neutral dye conjugates that are spectrally similar to the negatively charged fluorescein 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® FL dye has proven useful for studying ligand-receptor interaction by fluoresence polarization. In addition, BODIPY® FL dye has little or no spectral overlap with longer-wavelength dyes such as tetramethylrhodamine and Texas Red® dye, making it a useful green fluorophore for multicolor applications.