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.

Alexa Fluor™ 660 C2 Maleimide (Invitrogen™)

Alexa Fluor® 660 is a bright, far-red fluorescent dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 660 dye is water soluble and pH-insensitive from pH 4 to pH 10.

The maleimide derivative of Alexa Fluor® 660 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® 660 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® 660 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 668/697 nm
Extinction coefficient: 112,000 cm-1M-1
Molecular weight: ~1000

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™ 350 C5 Maleimide (Invitrogen™)

Alexa Fluor® 350 is a blue fluorescent dye with moderate photostability and excitation that matches the 350 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 350 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 350 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® 350 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® 350 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® 350 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 345/444 nm
Extinction coefficient: 17,000 cm-1M-1
Spectrally similar dyes: Marina Blue
Molecular weight: 578.68

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.

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.

Badan (6-Bromoacetyl-2-Dimethylaminonaphthalene) (Invitrogen™)

The thiol reactive badan 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 badan conjugates may make this useful for distinguishing thiols that are located in membranes versus those exposed to aqueous solvation in cells.

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.

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.

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.

DyLight™ 405 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 405 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 405 used to fluorescently label sulfhydryl-containing peptides, proteins and other biomolecular probes.

DyLight 405 has high fluorescence intensity over a broad pH range (pH 4-9) and is more photostable than Alexa Fluor™ 405, Cascade™ Blue 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 405 Maleimide:

High performance—DyLight 405 shows brighter fluorescence than Alexa Fluor 405, Cascade Blue 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 405 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.

EZ-Link™ Maleimide-PEG11-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Maleimide-PEG11-Biotin is a long, maleimide-activated, sulfhydryl-reactive biotinylation reagent that includes an 11-unit polyethylene glycol spacer arm for increased water-solubility and reach.

Features of EZ-Link Maleimide-PEG11-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)
Maleimide-activated—perform reactions at pH 6.5 to 7.5 in buffers such as PBS
Pegylated – spacer arm contains a hydrophilic, 11-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
Long—spacer arm (total length added to target) is 59.1 angstroms

Maleimide-PEG11-Biotin enables simple and efficient biotinylation of antibodies, cysteine-containing peptides and other thiol-containing molecules. The maleimide group reacts specifically and efficiently with reduced thiols (sulfhydryl groups,—SH) at pH 6.5 to 7.5 to form stable thioether bonds. The hydrophilic, 11-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. 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.

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.

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.

Alexa Fluor™ 750 C5 Maleimide (Invitrogen™)

Alexa Fluor® 750 is a bright, near-infrared fluorescent dye with excitation ideally suited for the 633 nm laser line or dye-pumped laser excitation. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 750 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 750 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® 750 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® 750 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® 750 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 753/783 nm
Extinction coefficient: 290,0000 cm-1M-1
Spectrally similar dyes: Cy7
Molecular weight: ~1350

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.

pHrodo™ Red Maleimide (Invitrogen™)

The thiol-reactive pH-sensitive pHrodo® Red Maleimide dye is suitable for the creation of bioconjugates to study endocytosis and phagocytosis. pHrodo® Red dramatically increases fluorescence as the pH of its surroundings become more acidic.
• Use pH-sensitive pHrodo® Red 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 green fluorescent markers such as GFP, pHrodo® Green, and many others

The increase in fluorescence of pHrodo® Red 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® Red makes it useful for multi-color experiments. pHrodo® Red 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® Red in a typical extracellular environment eliminates the need for wash steps or quencher dyes in the experimental workflow.

pHrodo® Red Maleimide is a thiol-reactive dye that can be used to create pHrodo® Red 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® Red is also available in an amine-reactive form (see pHrodo® Red SE), as well as a selection of ready-to-use conjugates (e.g., E. coli, S. aureus, and dextran). In addition, pHrodo® Green 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.

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.

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.

DyLight™ 488 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 488 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 488 used to fluorescently label sulfhydryl-containing peptides, proteins, and other biomolecular probes.

DyLight 488 has high fluorescence intensity over a broad pH range (pH 4-9) and is more photostable than Cy2™ dye, Alexa Fluor™ 488, FITC, and LI-COR™ dyes in many applications. The high water solubility of DyLight Fluors allows a high dye-to-protein ratio to be achieved without causing precipitation of the conjugates.

Features of DyLight 488 Maleimide:

High performance—DyLight 488 shows brighter fluorescence than Alexa Fluor 488, FITC and Cy2 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 488 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™ 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™ 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.

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.

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.

Alexa Fluor™ 680 C2 Maleimide (Invitrogen™)

Alexa Fluor® 680 is a bright, near-infrared fluorescent dye with excitation ideally suited for the 633 nm laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 680 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 680 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® 680 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® 680 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® 680 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 684/714 nm
Extinction coefficient: 175,000 cm-1M-1
Spectrally similar dyes: Cy5.5, IRDye 680LT
Molecular weight: ~1000

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.

QSY™ 9 C5-Maleimide (Invitrogen™)

The thiol-reactive quencher, QSY® 9 succinimidyl ester has a broad and intense visible absorption (~560 nm) but no fluorescence making it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications.

Oregon Green™ 488 Iodoacetamide, mixed isomers (Invitrogen™)

The thiol reactive Oregon Green® 488 iodoacetamide can be used to can be used to create green fluorescent bioconjugates with excitation/emission maxima ~496/524 nm.
This fluorinated analog of fluorescein overcomes some of the key limitations of fluorescein, including greater photostability and a lower pKa (pKa ~ 4.7 versus 6.4 for fluorescein), making its fluorescence essentially pH insensitive in the physiological pH range.

N-(1-Pyrene)Maleimide (Invitrogen™)

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

Alexa Fluor™ 647 C2 Maleimide (Invitrogen™)

Alexa Fluor® 647 is a bright, far-red fluorescent dye with excitation ideally suited for the 594 nm or 633 nm laser lines. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 647 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 647 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® 647 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® 647 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® 647 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 651/671 nm
Extinction coefficient: 265,000 cm-1M-1
Spectrally similar dyes: Cy5
Molecular weight: ~1250

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.

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

Thermo Scientific EZ-Link Maleimide-PEG2-Biotin is a mid-length, maleimide-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 Maleimide-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)
Maleimide-activated—perform reactions at pH 6.5 to 7.5 in buffers such as PBS
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 29.1 angstroms

Maleimide-PEG2-Biotin enables simple and efficient biotinylation of antibodies, cysteine-containing peptides and other thiol-containing molecules. The maleimide group reacts specifically and efficiently with reduced thiols (sulfhydryl groups,—SH) at pH 6.5 to 7.5 to form stable thioether bonds. 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. 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.

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

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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.

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.

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.

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.

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

Thermo Scientific EZ-Link Iodoacetyl-LC-Biotin is a mid-length, haloacetyl-activated, sulfhydryl-reactive biotinylation reagent that forms stable, irreversible thioether bonds at alkaline pH.

Features of EZ-Link Iodoacetyl-LC-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)
Iodoacetyl-activated—perform reactions in the dark at pH 7.5 to 8.5 in Tris or borate buffer
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 27.1 angstroms; contains hexylenediamine extension

Iodoacetyl-LC-Biotin is a haloacetyl-biotin compound for labeling protein cysteines and other molecules that contain sulfhydryl groups. This reagent specifically reacts with reduced thiols (-SH) in alkaline buffers to form permanent (irreversible) thioether bonds. The unique feature of Iodoacetyl-LC-Biotin is its extended yet chemically simple hexylenediamine spacer arm.

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.

Applications:
• Electron microscopy studies on spatial relationships between proteins (Ref.1)
• Localizing the SH1 thiol of the myosin head using avidin-biotin complexes in electron microscopy (Ref.2)

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.

Tris-(2-Carboxyethyl)phosphine, Hydrochloride (TCEP) (Invitrogen™)

Disufide crosslinks of cystines in proteins can be reduced to cysteine residues by TCEP (tris-(2-carboxyethyl)phosphine). Unlike DTT (dithiothreitol), TCEP does not contain thiols and therefore usually does not need to be removed prior to thiol modification.

6-IAF (6-Iodoacetamidofluorescein) (Invitrogen™)

The thiol-reactive 6-iodoacetamidofluorescein (6-IAF) can be used to produce bioconjugates with the 5-isomer of fluorescein.

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.

Texas Red™ C2 Maleimide (Invitrogen™)

The thiol-reactive Texas Red® C2 maleimide can be used to can be used to create bright red-fluorescent bioconjugates with excitation/emission maxima ~595/615 nm.

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.

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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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.

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.

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.

Monochlorobimane (mBCI) (Invitrogen™)

Monochloromobimane is essentially nonfluorescent until conjugated, readily reacts with several low molecular weight thiols, including glutathione, N-acetylcysteine, mercaptopurine, peptides and plasma thiols. The glutathione conjugate of monochlorobimane has absorption/emission maxima ~394/490 nm.

EZ-Link™ Maleimide Activated Horseradish Peroxidase (Thermo Scientific™)

Thermo Scientific Pierce Maleimide Activated Horseradish Peroxidase is for preparation of HRP conjugates with proteins, peptides or other ligands that contain sulfhydryl groups, such as reduced cysteines. This product contains 5 mg of conjugated protein and can effectively modify 5 mg of immunoglobulin.

Features of Maleimide Activated Horseradish Peroxidase:

Activated HRP – horseradish peroxidase (HRP) modified with maleimide groups for conjugation to sulfhydryl molecules
Sulfhydryl-reactivemaleimide groups conjugate to reduced thiols (-SH), as in the side-chain of cysteine residues
High activity HRP – enzyme activity is greater than 240 units/mg; lyophilized, activated enzyme is stable for at least 12 months at 4°C
Complete kit – includes the activated HRP as well as two types of reagents for sulfhydryl-ready antibodies (IgG) or proteins for conjugation

This product consists of horseradish peroxidase (HRP) that has been modified with Sulfo-SMCC (Part No. 22322) to attach several maleimide groups per HRP molecule while retaining the peroxidase activity. The activated HRP will covalently attach to proteins or other molecule containing sulfhydryl groups (e.g., cysteines). HRP-conjugates of antibodies, proteins, peptides and other thiol-containing reporter probes are easily made using this method. The complete kit includes the activated HRP as well as two types of reagents for preparing sulfhydryl-ready antibodies (IgG) or proteins for conjugation.

The complete kit for Maleimide Activated Horseradish Peroxidase contains reagents for exposing or added the necessary sulfhydryl groups on antibodies (IgG) or practically any other protein. These general strategies are described briefly in the applications section of our review of Maleimide Reaction Chemistry. Of course, any protein that contains cysteines has sulfhydryl groups (-SH), but they must be reduced (not in the form of disulfide bonds) to be conjugated. Antibodies also contain disulfide bonds that can be targeted as antibody labeling sites; the hinge-region disulfide bonds in IgG can be selectively cleaved with the mild reducing agent 2-Mercaptoethylamine (Part No. 20408), which is included in the complete kit. Alternatively, sulfhydryl groups can be added to proteins (or any amine-containing molecule) using SATA reagent (Part No. 26102), which also is included in the kit.

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EZ-Link™ Maleimide Activated Horseradish Peroxidase Kit