Shop All Thiol-Reactive Fluorophores, Biotins & Other Labels

Oregon Green™ 488 Maleimide (Invitrogen™)

The thiol reactive Oregon Green® 488 maleimide 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.

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.

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

Thermo Scientific Pierce Maleimide Activated Horseradish Peroxidase Kit is for preparation of HRP conjugates with proteins, peptides or other ligands that contain sulfhydryl groups, such as reduced cysteines. It is sufficient fo the conjugation of 5 mg of immunoglobulin (IgG). The kit contains all buffers and one 10 mL desalting column.

Features of the Maleimide Activated Horseradish Peroxidase Kit :

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.

Related Products
EZ-Link™ Maleimide Activated Horseradish Peroxidase

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.

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.

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™ 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.

DyLight™ 650-4xPEG Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 650-4xPEG Sulfhydryl-Reactive Dye is a maleimide-activated derivative of our high-performance DyLight 650 Dye used to fluorescently label cysteine-containing peptides, proteins, or other biomolecular probes.

The DyLight 650-4xPEG Dye contains 4 polyethylene glycol (PEG) chains that are non-toxic, enhance fluorescence, and reduce nonspecific binding of conjugates made with them. Conjugates made with DyLight 650-4xPEG Dye can be used as molecular probes for cellular and in vivo imaging, flow cytometry, IHC, and other fluorescence detection methods. 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 650-4xPEG Maleimide:

High fluorescence intensity—significantly brighter fluorescence than Alexa Fluor™ 647
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 650-4xPEG 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.

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.

Eosin-5-Maleimide (Invitrogen™)

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

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.

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.

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.

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)