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.

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.

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.

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.

Related Products
EZ-Link™ Maleimide Activated Horseradish Peroxidase Kit

5-IAF (5-Iodoacetamidofluorescein) (Invitrogen™)

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

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.

Monobromobimane (mBBr), 25 mg (Invitrogen™)

Monobromobimane 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 monobromobimane has absorption/emission maxima ~394/490 nm.

Fluorescein-5-Maleimide (Thermo Scientific™)

Thermo Scientific Fluorescein-5-Maleimide 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. Fluorescein-5-Maleimide is a sulfhydryl-reactive derivative of fluorescein that labels proteins and other molecules having free thiols (cysteine side chains)

Properties of Fluorescein-5-Maleimide:

• Alternative names: 5-MF, 5-maleimido-fluorescein
• Chemical name: 1H-Pyrrole-2,5-dione, 1-(3',6'-dihydroxy-3-oxospiro (isobenzofuran-1(3H),9'-(9H)xanthen-5-yl)-
• Molecular weight: 427.36±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 #: 75350-46-8
• Solubility: Soluble in DMF or DMSO
• Reactive groups: Maleimide, reacts with sulfhydryls at pH 6.5 to 7.5

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

Related Products
5-IAF (5-iodoacetamido-fluorescein)

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.

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.

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.

N-(1-Pyrene)Iodoacetamide (Invitrogen™)

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

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.

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.

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.