Shop All Thiol-Reactive Fluorophores, Biotins & Other Labels

4-Acetamido-4'-Maleimidylstilbene-2,2'-Disulfonic Acid, Disodium Salt (Invitrogen™)

Our 4-acetamido-4'-maleimidylstilbene-2,2'- disulfonic acid is a thiol-reactive reagent that is water soluble, with high polarity and membrane impermeability. This polysulfonated dye is useful for determining whether thiol-containing proteins and polypeptide chains are exposed at the extracellular or cytoplasmic membrane surface.

5-IAF (5-iodoacetamido-fluorescein) (Thermo Scientific™)

Thermo Scientific 5-Iodoacetamido-Fluorescein (5-IAF) is a high-performance derivative of fluorescein dye, activated for easy and reliable labeling of antibodies, proteins and other molecules for use as fluorescent probes. 5-Iodoacetamido-Fluorescein is a sulfhydryl-reactive derivative of fluorescein that labels proteins and other molecules having free thiols (cysteine side chains)

Properties of 5-Iodoacetamido-Fluorescein:

• Alternative names: 5-IAF, 5-iodoacetamidofluorescein
• Chemical name: Acetamide, N-(3',6'-dihydroxy-3-oxospiro (isobenzofuran-1(3H), 9'-(9H)xanthen)-5-yl)-2-iodo
• Molecular weight: 515.26±3
• Excitation source: 488 nm spectral line, argon-ion laser
• Excitation wavelength: 494 nm
• Emission wavelength: 518 nm
• Extinction coefficient: > 80,000 M-1cm-1
• CAS #: 63368-54-7
• Solubility: Soluble in DMF; aqueous buffers at pH > 6
• Reactive groups: Iodoacetamide, reacts with sulfhydryls at pH 7.0 to 7.5

Applications:
• Label antibodies for use as immunofluorescent probes
• Label oligonucleotides for hybridization probes
• Detect proteins in gels and on Western blots

Related Products
Fluorescein-5-Maleimide

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™ 650 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 650 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 650 used to fluorescently label sulfhydryl-containing peptides, proteins, and other biomolecular probes.

DyLight 650 provides vibrant far-red fluorescence with comparable or improved performance over other dyes, including Alexa Fluor™ 647 and Cy5™ dye, 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 650 Maleimide:

High performance—DyLight 650 fluoresces brighter than Alexa Fluor 647 and Cy5
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 650 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™ 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.

Rhodamine Red™ C2 Maleimide (Invitrogen™)

The thiol-reactive Rhodamine Red® C2 maleimide can be used to create red-fluorescent bioconjugates with excitation/emission maxima ~560/580 nm.

N-(Biotinoyl)-N'-(Iodoacetyl)Ethylenediamine (Invitrogen™)

The thiol-reactive biotin iodoacetamide reagent can be used to covalently attach biotin to thiol-containing proteins or thiolated nucleic acids.  The addition of a biotin residue enables the detection with avidin or streptavidin conjugates.  Electrophoretically separated thiol-containing proteins treated with biotin iodoacetamide have been detected in Western blots using an avidin–alkaline phosphatase conjugate.

Alexa Fluor™ 633 C5 Maleimide (Invitrogen™)

Alexa Fluor® 633 is a bright, red fluorescent dye with excitation ideally suited to the 633 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 633 dye is water soluble and pH-insensitive from pH 4 to pH 10.

The maleimide derivative of Alexa Fluor® 633 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® 633 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® 633 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 622/640 nm
Extinction coefficient: 143,000 cm-1M-1
Molecular weight: ~1300

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™ 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.

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

Monobromobimane (mBBr), FluoroPure™ grade (Invitrogen™)

Because it is manufactured at our ISO 9001—certified facilities in Eugene, Oregon, we can guarantee that FluoroPure grade monobromobimane is greater than or equal to 98% pure by HPLC.

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.

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.

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.

BODIPY™ FL Iodoacetamide (BODIPY™ FL C1-IA, N-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-yl)Methyl)Iodoacetamide) (Invitrogen™)

The thiol-reactive BODIPY® FL iodoacetamide can be used to create green-fluorescent bioconjugates. The electronically neutral BODIPY® FL dye has the most fluorescein-like spectra of the green-fluorescent BODIPY® dyes.