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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)

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™ Sulfo-NHS-LC-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Sulfo-NHS-LC-Biotin is an intermediate-length, water-soluble biotinylation reagent for labeling antibodies, proteins and other molecules that have primary amines.

Features of EZ-Link Sulfo-NHS-LC-Biotin:

Protein labeling—biotinylate antibodies to facilitate immobilization, purification or detection using streptavidin resins or probes
Cell surface labeling—biotinylates only surface proteins of whole cells because the negatively charged reagent does not permeate cell membranes
Amine-reactive—reacts with primary amines (-NH2), such as lysine side-chains, or the amino-termini of polypeptides
Soluble—charged sulfo-NHS group increases reagent water solubility compared to ordinary NHS-ester compounds
Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
Medium length—spacer arm (total length added to target) is 22.4 angstroms; provides excellent balance between adequate length (minimal steric hindrance for biotin binding) and modest mass

Sulfo-NHS-LC-Biotin is one of three very similar EZ-Link Reagents that are water-soluble, non-cleavable, and enable simple and efficient biotinylation of antibodies, proteins and any other primary amine-containing macromolecules in solution. Specific labeling of cell surface proteins is another common application for these uniquely water-soluble and membrane impermeable reagents. Differing only in their spacer arm lengths, the three Sulfo-NHS-ester reagents offer the possibility of optimizing labeling and detection experiments where steric hindrance of biotin binding is an important factor. Sulfo-NHS-LC-Biotin is offered in several package sizes and as complete protein labeling kits.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency, and performance for the intended research applications.

N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.

Related Products
EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit
EZ-Link™ Micro Sulfo-NHS-LC-Biotinylation Kit
Pierce™ Premium Grade Sulfo-NHS-LC-Biotin

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.

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

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

Alexa Fluor™ 514 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 514 is a bright, green-fluorescent dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 514 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 514 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.

The NHS ester (or succinimidyl ester) of Alexa Fluor® 514 is the most popular tool for conjugating this dye to a protein or antibody. NHS esters can be used to label to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting Alexa Fluor® conjugate will exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® NHS ester:

Fluorophore label: Alexa Fluor® 514 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 517/542 nm
Extinction coefficient: 80,000 cm-1M-2

Typical Conjugation Reaction
You can conjugate amine-reactive reagents with virtually any protein or peptide (the provided protocol is optimized for IgG antibodies). You can scale the reaction for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The Alexa Fluor® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO) (D12345), and the reaction is carried out in 0.1–0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

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.

SiteClick™ Qdot™ 565 Antibody Labeling Kit (Invitrogen™)

Create a perfectly labeled antibody with the SiteClick™ Qdot® 565 Antibody Labeling Kit. This kit replaces the conventional Qdot® 565 Antibody Conjugation Kit (Q22031MP). Unlike the conventional amine-thiol crosslinker method, SiteClick™ labeling specifically attaches the label to the heavy chains of an IgG antibody ensuring that the antigen binding domains remain available for binding to your antigen target. This site selectivity is achieved by targeting the carbohydrate domains present on essentially all IgG antibodies regardless of isotype and host species. In addition, no harsh reduction steps are required, and the labeling is consistent and reproducible each time it is performed. Depending upon the label, the resulting SiteClick™ labeled antibody can be used in flow cytometry, fluorescence imaging, or western blot detection.

Important Features of the SiteClick™ Qdot® 565 Antibody Labeling Kit:

• Contains everything required to label 100 µg of IgG antibody
• Easy to follow step-by-step protocol
• Highly efficient, site-specific, reproducible labeling chemistry results in high quality antibody conjugate.
• Qdot® 565 labels can be used in confocal or traditional fluoresce microscopy.
• In flow cytometry, Qdot® 565 can be excited by the 488 nm line of the argon-ion laser, or alternatively via excitation at 405 nm. This is true of all Qdot® fluorophores.

Qdot® Fluorophores Are Our Brightest Labels
Antibody conjugates made with Qdot® fluorophores produce fluorescence output that surpasses that of traditional organic dyes. Paired with the correct optical filters, Qdot® nanocrystals are as much as 50 times brighter. Read more about Qdot® nanocrystals or review additional product details in Qdot® Nanocrystals—Section 6.6 in the Molecular Probes® Handbook.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits (see Antibody Labeling from A to Z). To learn more about our various kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

Custom SiteClick™ Antibody Labeling Services
If you have an antibody that is considered "difficult to label" or has lost activity after labeling using a conventional method, please contact our custom service representatives to determine whether the SiteClick™ Antibody Labeling Service would be right for your antibody. We offer complete custom SiteClick™ antibody labeling services with the option of multiple detection molecules including biotin, Alexa Fluor® dyes, Qdot® fluorophores, R-PE, and others.

CellTracker™ Deep Red Dye (Invitrogen™)

CellTracker™ Deep Red is a fluorescent dye well suited for monitoring cell movement or location. After loading the cells, the dye is well retained, allowing for multigenerational tracking of cellular movements. The deep red excitation/emission spectra are ideal for multiplexing with green and red fluorescent dyes and proteins.

Need a different emission spectrum or longer tracking? View our other mammalian cell tracking products.

• Easy to use—remove medium, add dye, incubate 30 minutes, and image cells
• Fluorescent signal retention of >72 hours (typically three to six generations)
• Red excitation/emission spectra (630/650 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

The CellTracker™ Deep Red fluorescent dye has been designed to freely pass through cell membranes into cells, where it is transformed into cell-impermeant reaction products. CellTracker™ Deep Red dye is retained in living cells through several generations. The dye is transferred to daughter cells but not adjacent cells in a population. CellTracker™ Deep Red dye is designed to display fluorescence for at least 72 hours, and the dye exhibits ideal tracking properties: it is stable, nontoxic at working concentrations, well retained in cells, and brightly fluorescent at physiological pH. Additionally, the excitation and emission spectra of CellTracker™ Deep Red dye are well separated from GFP (green fluorescent protein) and RFP (red fluorescent protein) spectra allowing for multiplexing.

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

Thermo Scientific EZ-Link NHS-LC-Biotin is a long-chain, NHS-ester activated biotinylation reagent for labeling primary amines (e.g., protein lysines), whose membrane permeability enables it to be used for general intracellular labeling.

Features of EZ-Link NHS-LC-Biotin:

Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
Membrane-permeable—can be used to label inside cells (intracellular)
Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
Irreversible—forms permanent amide 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 22.4 angstroms; it consists of the biotin valeric acid group extended by a 6-atom chain

NHS-LC-Biotin is succinimidyl-6-(biotinamido)hexanoate. It is one of three similar EZ-Link NHS-Biotin Reagents that enable simple and efficient biotinylation of antibodies, proteins and any other primary amine-containing biomolecules in solution. Differing only in their spacer arm lengths, the three NHS-ester reagents offer researchers the possibility of optimizing labeling and detection experiments where steric hindrance of biotin binding is an important factor. Because they are uncharged and contain simple alkyl-chain spacer arms, these biotin compounds are membrane-permeable and useful for intracellular labeling.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.

pHrodo™ iFL Red STP Ester (amine-reactive) (Invitrogen™)

The amine-reactive pH-sensitive pHrodo iFL Red STP Ester dye is an improved version of pHrodo Red dye optimized for the creation of bioconjugates to be used in the study of antibody internalization, endocytosis, and phagocytosis. pHrodo iFL Red dye dramatically increases fluorescence as the pH of its surroundings become more acidic. It is more soluble than the original pHrodo Red dye, making it useful for the labeling of antibodies that may otherwise precipitate out of solution during conjugation.

• Use pH-sensitive pHrodo iFL Red STP Ester 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 iFL Green STP, and many others

The increase in fluorescence of pHrodo iFL Red dye as the pH changes from basic to acidic correlates with the acidification of intracellular vesicles, making it a valuable tool for the study of endocytosis or phagocytosis and their regulation by environmental factors, drugs, or pathogens. The spectral properties of pHrodo iFL Red dye make it useful for multi-color experiments. pHrodo iFL Red dye can be detected using most standard red-fluorescent filter sets and has been validated for use in a variety of applications, including flow cytometry, fluorescent microscopy, and high content screening (HCS). The lack of fluorescence of pHrodo iFL Red dye in a typical extracellular environment eliminates the need for wash steps or quencher dyes in the experimental workflow.

pHrodo iFL Red STP Ester is an amine reactive dye that can be used to make pHrodo iFL Red STP bioconjugates in aqueous buffer. The STP ester will react with primary amines on a protein, cell, or virus to create a stable conjugate that can be used in live cell assays or stored for later use.

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.

Zenon™ Alexa Fluor™ 488 Mouse IgG2a Labeling Kit (Invitrogen™)

Zenon® labeling technology provides a fast, versatile, and reliable method for adding a fluorescent label to an antibody. You need only a small amount of starting material, and the method is optimized for efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Antibody conjugates formed using Zenon® technology may be used in any protocol where a directly labeled primary antibody is suitable, including flow cytometry, imaging, and high-throughput applications. This exclusive Molecular Probes® Zenon® labeling technology greatly simplifies the use of multiple mouse-derived antibodies in the same staining protocol.

Important Features of Zenon® Labeling Technology:

• Labeled antibodies typically ready to use in 10 minutes
• Requires only 1–20 μg primary antibody
• Simple, no purification required
• Flexible–over 24 fluorophores plus biotin, HRP, alkaline phosphatase, and TSA to choose from
• Multiplex with other mouse monoclonal antibodies simultaneously


Save Time and Antibody
Each kit comes with affinity-purified monovalent Fab fragment of a goat anti-Fc antibody (or, in the case of the Zenon® Goat IgG Labeling Kits, a rabbit anti-Fc antibody) that has been conjugated to one of our premier Alexa Fluor® dyes or to Pacific Blue™, Pacific Orange™, fluorescein, or Texas Red®-X dyes, biotin R-phycoerythrin (R-PE), allophycocyanin (APC), HRP, or alkaline phosphatase.

Formation of the Fab–antibody complex with the Zenon® Antibody Labeling Kits is extremely fast (5 min for complex, 5 min for blocking step). And Zenon® labeling is a reliable and reproducible method, even with as low 0.4 μg in 2 μL of primary antibody. There is minimal waste of expensive or difficult-to-obtain antibodies when using the Zenon® Antibody Labeling Kits.

Preserve Primary Antibody Function and Affinities
Reactive dye labeling of primary antibodies can have unpredictable and undesirable outcomes. Among these are reduced binding affinities by label addition in the binding pocket. Zenon® antibody labeling approach, targeted to the Fc tail, avoids this concern.

Moreover the Zenon® dye- and enzyme-labeled Fab fragments have been affinity purified during their preparation to help ensure their high affinity and selectivity for the Fc portion of the corresponding primary antibody. The procedure for chemical labeling of the Fab fragments protects the Fc-binding site, resulting in more active labeling reagents.

Many Fluorophore and Enzyme Labels Available
Zenon® immunolabeling technology makes it very easy to change fluorescent color combinations or detection methodologies by simply using a different dye- or enzyme-labeled Fab fragment from our extensive selection of over 100 Zenon® Antibody Labeling Kits. If larger quantities or covalent attachment of the label is desired, see Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices.

Zenon® Technology Simplifies the Use of Multiple Antibodies of the Same Isotype in the Same Protocol
The stability of the Zenon® complex is sufficient to allow sequential (or simultaneous) labeling of different targets in cells and tissues with multiple antibody complexes. Subsequent to staining, an aldehyde-based fixation step can permanently block the transfer of Zenon® labels between different primary antibodies and will preserve the staining pattern.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody 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.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Related Links:

Zenon® Labeling Technology
Zenon® Technology: Versatile Reagents for Immunolabeling—Section 7.3

Oregon Green™ 488-X, Succinimidyl Ester, 6-isomer (Invitrogen™)

The amine-reactive Oregon Green® 488-X, succinimidyl ester 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. This reactive dye contains an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and the succinimidyl ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated.

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™ 615-B1 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 615-B1 NHS Ester is a benzopyrillium-based red-emitting specialty dye that can be used to label peptides, antibodies, and other proteins at primary amines. This dye has excitation and emission peaks at 623 and 643 nm, respectively (in ethanol) and an extinction coefficient of 200,000 M-1cm-1

General features of DyLight red-emitting specialty dyes:

Large selection—the largest family of dyes available for red-emitting fluorescence applications
NHS ester reactive group—allows immediate labeling of antibodies, proteins, peptides, and other amine-containing molecules through amide bond formation
Multiple solubility options—choose from hydrophilic to hydrophobic dyes to optimize the right dye label for the best performance in a given application

DyLight Red-emitting Dyes are a family of labeling agents that provide bright fluorescence detection for imaging. Dyes can be selected based upon their characteristic excitation and emission properties or relative hydrophilicity and hydrophobicity attributes. Dyes that contain a greater number of negatively charged sulfonates generally will have greater water solubility than dyes with fewer sulfonates. More hydrophobic dyes often provide better cell penetrating ability in vivo, while more hydrophilic dyes have less nonspecific binding potential. Each dye contains an amine-reactive NHS ester for simple modification of antibodies, proteins, peptides or other biomolecules through amide bond formation.

DyLight Red-emitting Dyes are a family of labeling agents that provide bright fluorescence detection for imaging. Dyes can be selected based upon their characteristic excitation and emission properties or relative hydrophilicity and hydrophobicity attributes. Dyes that contain a greater number of negatively charged sulfonates generally will have greater water solubility than dyes with fewer sulfonates. More hydrophobic dyes often provide better cell penetrating ability in vivo, while more hydrophilic dyes have less nonspecific binding potential. Each dye contains an amine-reactive NHS ester for simple modification of antibodies, proteins, peptides or other biomolecules through amide bond formation.

Criteria to consider when choosing a DyLight Red-emitting Specialty Dye
• Excitation and emission wavelengths—choose the best dye to match the excitation and emission capabilities of your instrument
• Water solubility—choose a dye based on its relative hydrophilicity, which directly correlates to the number of negatively-charged sulfonates it has on its core structure. More hydrophilic dyes are best at maintaining water solubility of a labeled antibody and limiting the nonspecific binding of the conjugate. More hydrophobic dyes often are best at penetrating tissues and cell membranes in vivo, meaning that dyes with fewer sulfonates may work best for some applications.
• DyLight dye selection—the broad selection of red-emitting dyes allows a number of candidate dyes to be tested in a given application for optimal performance.

Applications:
• Fluorescence imaging
• Confocal microscopy
• Flow cytometry
• Spectral fluorescence imaging
In vivo imaging
• Fluorescent western blotting
• Protein microarrays
• Antibody labeling
• Peptide labeling
• Fluorescence correlation spectroscopy
• Protein arrays
• Single molecule detection
• Nanoparticle conjugation
• Biotin/streptavidin conjugation

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