Shop All Thiol-Reactive Fluorophores, Biotins & Other Labels

Eosin-5-Maleimide (Invitrogen™)

Bioconjugates made with the thiol-reactive eosin-5-maleimide can be used as phosphorescent probes or as photosensitizers. With its high quantum yield (~0.57) for singlet oxygen generation, eosin and its conjugates can be used as effective photooxidizers of diaminobenzidine (DAB) in high-resolution electron microscopy studies and in correlated fluorescence and electron microscopy applications.

Pacific Blue™ C5-Maleimide (Invitrogen™)

Pacific Blue™ C5-maleimide is an excellent reagent for thiol-selective modification, quantitation and analysis. In this reaction, the thiol is added across the double bond of the maleimide to yield a thioether. This compound does not react with methionine, histidine or tyrosine. Reaction of Pacific Blue™ C5-maleimide with amines usually requires a higher pH than reaction of maleimides with thiols.

View all Pacific Blue™ dye products..

View the Fluorophore Selection Guide.

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.

N-(1-Pyrene)Maleimide (Invitrogen™)

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

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.

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.

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

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.

Click-IT™ Maleimide DIBO Alkyne, for copper free click chemistry (Invitrogen™)

Conjugates prepared with this thiol-reactive maleimide DIBO alkyne, can be detected with an azide-containing molecule in a copper-free 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 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, and with the elimination of copper, no detectable damage to cells or proteins. 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.

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.

DyLight™ 594 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 594 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 594 used to fluorescently label sulfhydryl-containing peptides, proteins and other biomolecular probes.

DyLight 594 provides vibrant orange-to-red fluorescence with better performance than other rhodamine derivatives including Alexa Fluor™ 594 and Texas Red™ 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 594 Maleimide:

High performance—DyLight 594 shows brighter fluorescence than Alexa Fluor 594 and Texas Red
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 594 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.

Iodoacetamide Alkyne (Invitrogen™)

Conjugates prepared with the thiol-reactive alkyne, succinimidyl ester can be detected with an azide-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.

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.

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.

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.