Shop All Thiol-Reactive Fluorophores, Biotins & Other Labels

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.

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.

DyLight™ 800 Maleimide (Thermo Scientific™)

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

DyLight 800 is a near-IR fluor that is invisible to the naked eye but increases the staining options when using infrared imaging systems. DyLight 800 has spectral properties that are very similar to other near-IR dyes, including Alexa Fluor™ 790 and IRDye™ 800. 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 800 Maleimide:

High performance—DyLight 800 replaces Alexa Fluor 800 and IRDye 800 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 800 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.

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DyLight™ 350 Maleimide

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.

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.

Alexa Fluor™ 568 C5 Maleimide (Invitrogen™)

Alexa Fluor® 568 is a bright, orange/red fluorescent dye with excitation ideally suited for the 568 nm laser line on the Ar-Kr mixed-gas laser. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 568 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 568 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® 568 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® 568 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® 568 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 575/600 nm
Extinction coefficient: 92,000 cm-1M-1
Spectrally similar dyes: Rhodamine red
Molecular weight: 880.92

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.

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.

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.

Iodoacetamide Azide (Invitrogen™)

Conjugates prepared with the thiol-reactive azide, succinimidyl ester can be detected with an alkyne-containing molecule in a click chemistry reaction. Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Click reactions have several characteristics: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; the components of the reaction are bioinert; and perhaps most importantly, no side reactions occur – the label and detection tags react selectively and specifically with one another. Unlike traditional chemical reactions utilizing succinimidyl esters or maleimides that target amines and sulfhydryls – functional groups that are not unique – click chemistry-labeled molecules can be applied to complex biological samples and be detected with unprecedented sensitivity due to extremely low background.

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.

Measure-IT™ Thiol Assay Kit (Invitrogen™)

The Measure-iT™ Thiol Assay Kit proves easy and accurate quantitation of thiol. The assay has a linear range of 0.05-5 uM thiol, making it up to 400 times more sensitive than colorimetric methods based on Ellman's reagent. The assay is performed at room temperature; maximum fluorescence signal is attained in 5 minutes and is stable for at least 1 hour.

BODIPY™ FL L-Cystine (Invitrogen™)

We have attached two BODIPY FL fluorophores to the amino groups of the disulfide-containing amino acid cystine to create this reagent for reversible thiol-specific labeling of thiolated oligonucleotides, proteins and cells. BODIPY FL L-cystine is virtually nonfluorescent due to interactions between the two fluorophores; however, thiol-specific exchange with thiolated biomolecules occurs to form mixed disulfides, resulting in green fluorescence.

Pierce™ Streptavidin, Hydrazide-Activated (Thermo Scientific™)

Thermo Scientific Pierce Hydrazide-Activated Streptavidin conjugate include recombinant streptavidin in a purified form activated for crosslinking to carbonyl groups in a molecule.

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Pierce™ Streptavidin, Alkaline Phosphatase Conjugated
Pierce™ Streptavidin, Maleimide-Activated

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.