Shop All Labels & Labeling Kits

CellTracker™ Violet BMQC Dye (Invitrogen™)

CellTracker™ Violet BMQC is a fluorescent dye well suited for monitoring cell movement or location. After loading into cells, the dye is well retained, allowing for multigenerational tracking of cellular movements. The violet 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)
• Violet excitation/emission spectra (415/516 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

The CellTracker™ Violet BMQC fluorescent dye has been designed to freely pass through cell membranes into cells, where it is is transformed into cell-impermeant reaction products. CellTracker™ Violet BMQC dye is retained in living cells through several generations. The dye is transferred to daughter cells but not adjacent cells in a population. CellTracker™ Violet BMQC 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™ Violet BMQC dye are well separated from GFP (green fluorescent protein) and RFP (red fluorescent protein) spectra allowing for multiplexing.

Click-IT™ AHA (L-Azidohomoalanine) (Invitrogen™)

Click-iT® AHA (L-azidohomoalaine) provides a fast, sensitive, non-toxic and most importantly non-radioactive alternative to the traditional radioactive technique, 35S-methionine for the detection of nascent protein. AHA is an amino acid analog that contains a very small modification, specifically an azido moiety that can be fed to cultured cells and incorporated into proteins during active protein synthesis. Detection utilizes the chemoselective ligation or “click " reaction between an azide and an alkyne where the azido modified protein is detected with one of the Click-iT® Protein Analysis Detection Kits containing either TAMRA, Dapoxyl®, or biotin alkyne. Detection sensitivity with these reagents in 1-D gels and Western blots is in the low femtomole range and compatible with downstream LC-MS⁄MS and MALDI MS analysis or Multiplexed Proteomics® reagents for differential analyses of newly synthesized proteins together with total glycoproteins, total phosphoproteins or total protein.

Click-IT™ L-Homopropargylglycine (HPG) (Invitrogen™)

Click-iT® homopropargylglycine (HPG) provides a fast, sensitive, non-toxic and most importantly non-radioactive alternative to the traditional technique,35S methionine for the detection of nascent protein synthesis. HPG is an amino acid analog that contains a very small modification, specifically an alkyne moiety that can be fed to cultured cells and incorporated into proteins during active protein synthesis. Detection utilizes the chemoselective ligation or “click" reaction between and azide and an alkyne where the alkyne modified protein is detected with an azide-containing dye or hapten together with either the Click-iT® Cell Reaction Buffer Kit or the Click-iT® Protein Buffer Kit. With the Click-iT® Cell Reaction Buffer Kit, cells can be analyzed by fluorescence microscopy, flow cytometry or high-content imaging and analysis. With the Click-iT® Protein Reaction Buffer Kit, achieve detection sensitivity in 1-D gels and western blots in the low femtomole range or perform LC-MS⁄MS and MALDI MS analysis.

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.

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

SiteClick™ Qdot™ 585 Antibody Labeling Kit (Invitrogen™)

Create a perfectly labeled antibody with the SiteClick™ Qdot® 585 Antibody Labeling Kit. This kit replaces the conventional Qdot® 585 Antibody Conjugation Kit (Q22011MP). 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® 585 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® 585 labels can be used in confocal or traditional fluoresce microscopy.
• In flow cytometry, Qdot® 585 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.

Alexa Fluor™ 532 Protein Labeling Kit (Invitrogen™)

Molecular Probes™ Protein Labeling Kits provide a convenient means for attaching a fluorescent label (or biotin) to an antibody (or a protein larger than 40 kDa). Conjugates are ideal for multiple applications, including flow cytometry, fluorescent microscopy, immunohistochemistry, primary detection, ELISAs, immunocytochemistry, FISH, and more. Kits are available in 12 Alexa Fluor™ dye colors, biotin, the hapten Oregon Green™ 488, fluorescein EX, and Texas Red™ dye. Each kit provides the components needed to perform three protein conjugations and purifications.

View a selection guide for all Protein Labeling Kits.

View the Fluorophore Selection Guide.

Important features of protein labeling kits:

• Labeled proteins typically ready to use in 2 hr (~30 min hands-on time)
• Designed to label 1 mg of IgG
• Simple protocol—react, separate, use
• Stabilizing proteins must be removed from the sample before labeling

The benefits of Alexa Fluor™ dyes
Compared to traditional dyes, Alexa Fluor™ dyes are brighter, more photostable, and more pH resistant between pH 4 and 10. And generally when using Alexa Fluor™ dyes, higher degrees of labeling can be achieved without intramolecular quenching.

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 Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes™ Handbook.

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.

QSY™ 9 Carboxylic Acid, Succinimidyl Ester (Invitrogen™)

The amine-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.

BODIPY™ 558/568 NHS Ester (Succinimidyl Ester) (Invitrogen™)

BODIPY® 558/568 dye is bright, red fluorescent dye. It has a high extinction coefficient and fluorescence quantum yield and is relatively insensitive to solvent polarity and pH change. In contrast to the highly water soluble fluorophores Alexa Fluor® 488 dye and fluorescein (FITC), BODIPY® dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds. BODIPY® 558/568 dye has a relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization-based assays and a large two-photon cross-section for multiphoton excitation. In addition to reactive dye formulations, we offer BODIPY® 558/568 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 BODIPY® 558/568 is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY® 558/568 conjugates exhibit bright fluorescence, narrow emission bandwidths, and relatively long excited-state lifetimes, which can be useful for fluorescence polarization assays and two-photon excitation (TPE) microscopy.

This reactive dye contains a C3 alkyl spacer between the fluorophore and the NHS 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.

Detailed information about this BODIPY® 558/568 NHS ester:

Fluorophore label: BODIPY® 558/568 dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 559/568 nm
Extinction coefficient: 97,000 cm-1M-1
Molecular weight: 443.23

Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. The reaction can be scaled 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 BODIPY® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), 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 BODIPY® 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 medium 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.

CellVue™ NIR815 Cell Labeling Kit (Invitrogen™)

CellVue™ dyes are lipophilic dyes that can be used to label the cell membrane for the purpose of identifying and tracking labeled cells. Cell labeling is rapid and stable and can be combined with fluorescently labeled antibodies and other markers of cellular function for flow cytometric analysis and fluorescent microscopy. Mini CellVue™ Kits are supplied with one vial of dye stock (1 mM in ethanol) and one vial of labeling vehicle (Diluent C).

CellVue™ NIR815 is a near-infrared fluorescent cell labeling reagent. It is optimally excited at 786 nm and has a peak emission of 814 nm. CellVue™ NIR815 has been reported to be useful for short-term in vivo cell tracking studies using a Pearl™ Imager or Odyssey Infrared Imaging System (Li-COR Biosciences, Inc., Lincoln, NE).

Reported Application
Microscopy, Immunocytochemistry, Cell Labeling

5-FAM (5-Carboxyfluorescein), single isomer (Invitrogen™)

The single isomer, 5-FAM, contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. Fluorescein is the most common fluorescent derivatization reagent for labeling biomolecules. In addition to its relatively high absorptivity, excellent fluorescence quantum yield, and good water solubility, fluorescein has an excitation maximum that closely matches the 488 nm spectral line of the argon-ion laser.

Alexa Fluor™ 647 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 647 is a bright and photostable far-red dye with excitation ideally suited to the 633 nm laser line. 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. Fluorescence of this long-wavelength Alexa Fluor® dye is not visible to the human eye but is readily detected by most imaging systems. 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 NHS ester (or succinimidyl ester) of Alexa Fluor® 647 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® 647 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 651/672 nm
Extinction coefficient: 270,000 cm-1M-1
Spectrally similar dyes: Cy5®
Molecular weight: ~1250

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.

EZ-Link™ Plus Activated Peroxidase (Thermo Scientific™)

Thermo Scientific Pierce Plus Activated Peroxidase is an amine-reactive form of horseradish peroxidase (HRP) that provides coupling efficiencies of greater than 95% with antibodies and other proteins. This product consists of one vial, containing 1 mg of lyophilized HRP and is sufficient to modify 1 mg of Immunoglobulin (IgG)

Features of Thermo Scientific Pierce Plus Activated Peroxidase:

Activated HRP – periodate-treated, aldehyde-activated horseradish peroxidase, ready for conjugation to antibodies and other proteins at sites of primary amines (e.g., lysines)
Permanent conjugation – reacts efficiently (95%) with primary amines to form covalent amide bonds upon treatment with sodium cyanoborohydride (included in kit)
High activity HRP – enzyme activity is 120 to 200 units/mg; lyophilized, activated enzyme is stable for at least 12 months at -20°C
Convenient quantities – each 1 mg-quantity of activated enzyme is sufficient for reaction with 1 mg of IgG to produce about 0.5 mL of conjugate
Customizable – vary the molar ratios, reaction buffer and pH, and other parameters to acheive conjugates with different levels of HRP incorporation and activity

Plus Activated Peroxidase is horseradish peroxidase (HRP) enzyme, whose native carbohydrates (sugars) have been gently oxidized with periodate to produce amine-reactive aldehyde groups. These carbonyls of Plus Activated Peroxidase spontaneously and efficiently crosslink with primary amines on antibody or other proteins. The method is more effective than other amine-reactive chemistries, such as glutaraldehyde coupling, which often causes polymerization and a greater degree of conjugate inactivation. The pre-activated enzyme eliminates the difficulties inherent in preparing and validating activated peroxidase from scratch. The kit provides the enzyme, accessory reagents and protocol to easily produce high-performance conjugates for Western blotting, ELISA and other detection techniques.

Related Products
EZ-Link™ Plus Activated Peroxidase Kit

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.

Zenon™ Alexa Fluor™ 532 Rabbit IgG 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

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.

Sulfo-SHPP (Water-Soluble Bolton-Hunter Reagent) (Thermo Scientific™)

Thermo Scientific Pierce Sulfo-SHPP, sulfosuccinimidyl-3 -(4-hydroxypheynyl) propionate, also known as water-soluble Bolton-Hunter Reagent, conjugates tyrosine-like functional groups to primary amines to increase the number of tyrosyl groups that can be iodinated by iodine-125 labeling procedures.

Features of Sulfo-SHPP:

• Iodinate before or after coupling Sulfo-SHPP to the molecule of interest
• Useful for introducing tyrosyl groups on proteins sensitive to small amounts of organic solvent
• Useful for labelling cell surfaces without exposing the cells to membrane permeable solvents

Click-IT™ Alexa Fluor™ 594 DIBO Alkyne, for copper free click chemistry detection of azide (Invitrogen™)

The fluorescent Alexa Fluor® 594 Dibo alkyne is reactive with azides via a copper-free "click chemistry" reaction.

Please consider this copper-free variation to our copper requiring alkynes if you are staining the surface of live cells or have concerns about native protein function loss with copper in cell extracts. Copper can damage fluorescent proteins, Quantum Dot nanocyrstals, certain enzymes and photoproteins like RPE. The DIBO reagent is not suitable for staining intracellular components of fixed and permeabilized cells due to high backgrounds.

SiteClick™ Qdot™ 625 Antibody Labeling Kit (Invitrogen™)

Create a perfectly labeled antibody with the SiteClick™ Qdot® 625 Antibody Labeling Kit. This kit replaces the conventional Qdot® 625 Antibody Conjugation Kit (A10197). 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® 625 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® 625 labels can be used in confocal or traditional fluorescence microscopy.
• In flow cytometry, Qdot® 625 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.

Alexa Fluor™ 546 Protein Labeling Kit (Invitrogen™)

Molecular Probes® Protein Labeling Kits provide a convenient means for attaching a fluorescent label (or biotin) to an antibody (or a protein larger than 40 kDa). Conjugates are ideal for multiple applications, including flow cytometry, fluorescent microscopy, immunohistochemistry, primary detection, ELISAs, immunocytochemistry, FISH, and more. Kits are available in 12 Alexa Fluor® dye colors, biotin, the hapten Oregon Green® 488, fluorescein EX, and Texas Red® dye. Each kit provides the components needed to perform three protein conjugations and purifications.

Important Features of Protein Labeling Kits:

• Labeled proteins typically ready to use in 2 hr (~30 min hands-on time)
• Designed to label 1 mg of IgG
• Simple protocol — react, separate, use
• Stabilizing proteins must be removed from the sample before labeling


The Benefits of Alexa Fluor® Dyes
Compared to traditional dyes, Alexa Fluor® dyes are brighter, more photostable, and more pH resistant between pH 4 and 10. And generally when using Alexa Fluor® dyes, higher degrees of labeling can be achieved without intramolecular quenching. For details see Alexa Fluor® Dyes Spanning the Visible and Infrared Spectrum—Section 1.3.

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 Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

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.

Qdot™ 605 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 605 ITK™ carboxyl quantum dots are the ideal starting material for preparing custom conjugates that require high loading of biomolecules. These materials are carboxylate functionalized and can be coupled to amine groups of proteins and modified oligonucleotides using EDC-mediated condensation. The coatings of these probes provides more binding sites than our Qdot® ITK™ amino quantum dots, but lacks PEG linkers that help to prevent non-specific interactions. These materials can be conjugated to X-PEG-amine bi-functional linkers for custom reactivity and higher specificity. Our Qdot® ITK™ carboxyl quantum dots are provided as 8 µM solutions and are available in all 9 Qdot® probe colors.

Important Features of Qdot® ITK™ Carboxyl Quantum Dots:
• Qdot® 605 ITK™ carboxyl quantum dot has emission maxima of ~605 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large Stokes shift
• Available in multiple colors
• Ideal labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the carboxyl-derivatized form, we offer Qdot® ITK™ quantum dots with amino and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

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

QSY™ 21 Carboxylic Acid, Succinimidyl Ester (Invitrogen™)

The amine-reactive quencher, QSY® 21 succinimidyl ester has a broad and intense absorption (~661 nm maxima) but no fluorescence making it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications.

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.

Probenecid, Water Soluble (Invitrogen™)

Probenecid is commonly used to inhibit organic-anion transporters located in the cell membrane. Such transporters can extrude dyes and indicators and thus contribute to poor loading or a high background signal in assays based on retention of the dyes or indicators inside cells. The use of probenecid to block the efflux of intracellular dyes was first described by Di Virgilio et al. (1990), and it has been used with a wide range of anionic dyes and conjugates. The commonly used free acid form of probenecid is difficult to dissolve, requiring 1 M NaOH to get it into solution. Our water-soluble probenecid (P36400) dissolves quickly in assay buffer and eliminates the need to handle caustic NaOH. The water-soluble probenecid is included in the Fluo-4 NW Calcium Assay Kits (F36205, F36206) and is also available separately.

BODIPY™ 576/589 NHS Ester (Succinimidyl Ester) (Invitrogen™)

BODIPY® 576/589 dye is bright, red fluorescent dye with similar excitation and emission to Rhodamine Red™ and Alexa Fluor®568. It has a high extinction coefficient and fluorescence quantum yield and is relatively insensitive to solvent polarity and pH change. In contrast to the highly water soluble fluorophores Alexa Fluor® 488 dye and fluorescein (FITC), BODIPY® dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds. BODIPY® 576/589 dye has a relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization-based assays and a large two-photon cross-section for multiphoton excitation. In addition to reactive dye formulations, we offer BODIPY® 576/589 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 BODIPY® 576/589 is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY® 576/589 conjugates exhibit bright fluorescence, narrow emission bandwidths, and relatively long excited-state lifetimes, which can be useful for fluorescence polarization assays and two-photon excitation (TPE) microscopy.

This reactive dye contains a C3 alkyl spacer between the fluorophore and the NHS 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.

Detailed information about this BODIPY® 576/589 NHS ester:

Fluorophore label: BODIPY® 576/589 dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 575/588 nm
Extinction coefficient: 83,000 cm-1M-1
Molecular weight: 426.19

Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. The reaction can be scaled 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 BODIPY® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), 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 BODIPY® 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 medium 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™ 488 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 488 is a bright, green-fluorescent dye with excitation ideally suited for 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 NHS ester (or succinimidyl ester) of Alexa Fluor® 488 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® 488 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 494/517 nm
Extinction coefficient: 73,000 cm-1M-1
Spectrally similar dyes: FITC, Oregon Green 488
Molecular weight: 643.4

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.

N-Methylisatoic Anhydride, high purity (Invitrogen™)

The amine-reactive N-methylisatoic anhydride can be used to create fluorescent bioconjugates with absorption/emission maxima ~316/386 nm.

CellVue™ NIR780 Cell Labeling Kit (Invitrogen™)

CellVue™ dyes are lipophilic dyes that can be used to label the cell membrane for the purpose of identifying and tracking labeled cells. Cell labeling is rapid and stable and can be combined with fluorescently labeled antibodies and other markers of cellular function for flow cytometric analysis and fluorescent microscopy. Mini CellVue™ Kits are supplied with one vial of dye stock (1 mM in ethanol) and one vial of labeling vehicle (Diluent C); Midi CellVue™ Kits are supplied with two vials of dye stock (1 mM in ethanol) and six vials of Diluent C.

CellVue™ NIR780 is a near-infrared fluorescent cell labeling reagent. It can be excited off the red laser line (633 nm) and has a peak emission of 776 nm that can be detected in filter sets designed for APC-eFluor™ 780. CellVue™ NIR780 is compatible with most multi-color applications, however, due to similar emission properties it cannot be used in combination with APC-eFluor™ 780 or APC-Alexa Fluor™ 750 reagents.

Reported Application
Flow Cytometric Analysis, Microscopy, Immunocytochemistry, Cell Labeling

BODIPY™ FL Hydrazide (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid, Hydrazide) (Invitrogen™)

BODIPY® FL hydrazide can be reversibly coupled to aldehydes and ketones to form a Schiff base - which can be reduced to a generate stable amine derivative by sodium borohydride (NaBH4) or sodium cyanoborohydride (NaCNH3). Carboxylic acids of proteins and other water-soluble biopolymers can be coupled to this molecule in aqueous solution using water-soluble carbodiimides such as EDAC (E2247).

Click-iT™ Plus OPP Alexa Fluor™ 488 Protein Synthesis Assay Kit (Invitrogen™)

The Click-iT® Plus OPP Alexa Fluor® 488 Protein Synthesis Assay Kit provides a fast, sensitive, and non-radioactive method for the detection of protein synthesis using fluorescence microscopy or high-content imaging. In this assay O-propargyl-puromycin (OPP) is efficiently incorporated into newly translated proteins in complete methionine-containing media and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific, and mild click reaction.

Features of the kit include:

• No media change required—works in complete, methionine-containing media, no need to remove cell media
• Multiplex-enabled—Click-iT® Plus technology retains signal from GFP and binding of fluorescent-conjugated phalloidins
• Non-radioactive—an alternative to the traditional 35S-methionine methods
• Works in vivo—published results demonstrate use in vivo for determination of protein translation

The kit contains O-propargyl-puromycin (OPP), which is an alkyne analog of puromycin (also available separately), as well as Alexa Fluor® 488 picolyl azide and all necessary reagents to perform the Click reaction. The OPP is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of the Alexa Fluor® 488 picolyl azide and the Click reaction reagents leads to a chemoselective ligation, or "click" reaction, between the picolyl azide dye and the OPP alkyne, allowing the modified proteins to be detected by imaged-based analysis.

The click reaction uses bioorthogonal (biologically unique) moieties to fluorescently label proliferating cells, helping produce low backgrounds and high detection sensitivities. Because of the mild reaction conditions, Click-iT® Plus assays detect protein translation events while enabling the preservation of cell morphology, the binding of fluorescently-labeled phalloidin, and the fluorescent signal from GFP.

Unlike 35S-methionine, used in traditional methods, OPP is not an amino acid analog, so it can be added directly to cells in complete media or used to determine protein synthesis in vivo.

The kit contains all of the components needed to label and detect the incorporated OPP in newly translated proteins in samples of adherent cells. The kit includes sufficient reagents for the labeling of 25 18 mm × 18 mm coverslips using 1 mL of reaction buffer per test.

Alexa Fluor™ 680 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 680 is a bright and photostable near-IR dye that is spectrally similar to the Cy5.5 dye. 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. Fluorescence of this long-wavelength Alexa Fluor® dye is not visible to the human eye but is readily detected by most imaging systems. 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 NHS ester (or succinimidyl ester) of Alexa Fluor® 680 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® 680 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 684/707 nm
Extinction coefficient: 183,000 cm-1M-1
Spectrally similar dyes: Cy5.5

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.

EZ-Link™ Micro Sulfo-NHS-Biotinylation Kit (Thermo Scientific™)

The Thermo Scientific EZ-Link Micro Sulfo-NHS-Biotinylation Kit contains reagents sufficient for 8 biotinylation reactions (e.g., 0.05–0.2 mg antibody per reaction). The EZ-Link Sulfo-NHS-Biotin reagent included in the kit is a short-chain, water-soluble biotinylation reagent used for labeling antibodies, proteins and other molecules that have primary amines.

Features of EZ-Link Sulfo-NHS-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
Very short—spacer arm (total length added to target) is 13.5 angstroms; it consists of the native biotin valeric acid group only.

Sulfo-NHS-Biotin is the shortest 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-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-Biotin
EZ-Link™ Sulfo-NHS-Biotinylation Kit

Zenon™ Alexa Fluor™ 546 Rabbit IgG 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

GlcNAz (N-azidoacetylglucosamine tetraacylated) (Thermo Scientific™)

Thermo Scientific Pierce GlcNAz (N-azidoacetylglucosamine-tetraacylated) is an azide-labeled sugar that provides a highly specific approach for studying glycoproteins through in vivo metabolic labeling and chemoselective ligation.

Features of Azido-Sugars:

Bioorthogonal—the azido group is small, nonreactive and absent from living systems; as such the azido-sugar compounds do not interfere with endogenous cellular pathways and substitute for their naturally occurring analogs
Compatible—reaction chemistry with phosphine compounds occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
Chemoselective—azide and phosphine groups do not react or interfere with components of biological samples but conjugate to one another with high efficiency
Versatile—azide tag can be targeted for detection, immobilization, conjugation or affinity purification depending on which phosphine-activated compound it is reacted with

These sugars are azide-derivatives of naturally occurring monosaccharides that cells use to glycosylate proteins using post-translational modification biochemical pathways. The azide functional group is small and nonreactive with endogenous molecules. When supplied to cells, these compounds become incorporated by glycosylation events to effectively "tag" glycoproteins with the azide group. The azide group then can be specifically targeted for detection or conjugation using alkyne-activated reagents ("click" chemistry) or phosphine-activated reagents (Staudinger ligation).

When used in combination with phosphine-activated fluorescent dyes, biotin reagents, and or other compounds, these azido-modified sugars facilitate the investigation of cellular pathways involving glycosylation.

There are several classes of glycoproteins grouped by the type of carbohydrate and amino acid linkage site. N-linked glycosylation is a modification of asparagine amines, whereas O-linked glycosylation occurs through the hydroxyl of serine and threonine residues. The azido-modified sugars are metabolic substitutes for endogenous amino sugars. ManNAz is converted by cells to an azido sialic acid derivative that is used for N-linked glycosylation of cell surface proteins. GlcNAz and GalNAz are predominantly used to label the O-linked glycosylation (O-GlcNAc and O-GalNAc).

Related Products
ManNAz (N-azidoacetylmannosamine tetraacylated)
GalNAz (N-azidoacetylgalactosamine tetraacylated)

Influx™ Pinocytic Cell-Loading Reagent (Invitrogen™)

Influx pinocytic cell-loading reagent provides a convenient, rapid and simple procedure for loading water-soluble materials into live cells. With the Influx reagent, polar compounds can be introduced into many cells simultaneously without significantly altering normal cell function.

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.

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.

SiteClick™ Qdot™ 655 Antibody Labeling Kit (Invitrogen™)

Create a perfectly labeled antibody with the SiteClick™ Qdot® 655 Antibody Labeling Kit. This kit replaces the conventional Qdot® 655 Antibody Conjugation Kit (Q22021MP). 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® 655 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® 655 labels can be used in confocal or traditional fluorescence microscopy.
• In flow cytometry, Qdot® 655 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.

Alexa Fluor™ 568 Protein Labeling Kit (Invitrogen™)

Molecular Probes® Protein Labeling Kits provide a convenient means for attaching a fluorescent label (or biotin) to an antibody (or a protein larger than 40 kDa). Conjugates are ideal for multiple applications, including flow cytometry, fluorescent microscopy, immunohistochemistry, primary detection, ELISAs, immunocytochemistry, FISH, and more. Kits are available in 12 Alexa Fluor® dye colors, biotin, the hapten Oregon Green® 488, fluorescein EX, and Texas Red® dye. Each kit provides the components needed to perform three protein conjugations and purifications.

Important Features of Protein Labeling Kits:

• Labeled proteins typically ready to use in 2 hr (~30 min hands-on time)
• Designed to label 1 mg of IgG
• Simple protocol — react, separate, use
• Stabilizing proteins must be removed from the sample before labeling


The Benefits of Alexa Fluor® Dyes
Compared to traditional dyes, Alexa Fluor® dyes are brighter, more photostable, and more pH resistant between pH 4 and 10. And generally when using Alexa Fluor® dyes, higher degrees of labeling can be achieved without intramolecular quenching. For details see Alexa Fluor® Dyes Spanning the Visible and Infrared Spectrum—Section 1.3.

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 Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

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.

Qdot™ 585 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 585 ITK™ carboxyl quantum dots are the ideal starting material for preparing custom conjugates that require high loading of biomolecules. These materials are carboxylate functionalized and can be coupled to amine groups of proteins and modified oligonucleotides using EDC-mediated condensation. The coatings of these probes provides more binding sites than our Qdot® ITK™ amino quantum dots, but lacks PEG linkers that help to prevent non-specific interactions. These materials can be conjugated to X-PEG-amine bi-functional linkers for custom reactivity and higher specificity. Our Qdot® ITK™ carboxyl quantum dots are provided as 8 µM solutions and are available in all 9 Qdot® probe colors.

Important Features of Qdot® ITK™ Carboxyl Quantum Dots:
• Qdot® 585 ITK™ carboxyl quantum dot has emission maxima of ~585 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large Stokes shift
• Available in multiple colors
• Ideal labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the carboxyl-derivatized form, we offer Qdot® ITK™ quantum dots with amino and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

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

DyLight™ 594 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 594 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 594 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

DyLight 594 provides vibrant red fluorescence with better performance than Alexa Fluor™ 594 and Texas Red™ dye for fluorescent 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. DyLight 594 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of DyLight 594 NHS Ester:

High performance—DyLight 594 shows brighter fluorescence than Alexa Fluor 594 and Texas Red
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications:
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 594 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require DyLight 594 Amine-Reactive Dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 594 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

We also offer Standard and Microscale DyLight 594 Antibody Labeling Kits for fast and efficient fluorescent labeling of antibodies for use in fluorescence methods. The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG. Both kit sizes include the Amine-Reactive DyLight 594 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

Related Products
DyLight™ 594 Antibody Labeling Kit
DyLight™ 594 Microscale Antibody Labeling Kit

Qtracker™ 625 Cell Labeling Kit (Invitrogen™)

The reagents in the Qtracker® 625 Cell Labeling Kit use a custom targeting peptide to deliver orange/red-fluorescent Qdot® 625 nanocrystals into the cytoplasm of live cells. Qtracker® Cell Labeling Kits are designed for loading cells grown in culture with highly fluorescent Qdot® nanocrystals. Once inside the cells, Qtracker® labels provide intense, stable fluorescence that can be traced through several generations, and are not transferred to adjacent cells in a population.

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

Key Attributes:

Qtracker® 625 label has Ex/Em (405-585/625) nm
Designed for loading cells grown in culture with highly fluorescent Qdot® nanocrystals
Provide intense, stable fluorescence that can be traced through several generations
Available in eight colors—525 nm, 565 nm, 585 nm, 605 nm, 625 nm, 655 nm, 705 nm, or 800 nm emission
Excellent tools for long-term tracking or imaging studies of live cells, including migration, motility, morphology, and other cell function assays

The Qtracker® Cell Labeling Kits use a custom targeting peptide to deliver Qdot® nanocrystals into the cytoplasm of live cells. Cytoplasmic delivery by this mechanism is not mediated by a specific enzyme; therefore, no cell-type specificity has been observed. Delivery is typically accomplished in less than 1 hour.

Qdot® nanocrystals delivered by the Qtracker® Cell Labeling Kits are compatible with serum-sensitive cells; intense fluorescence is maintained in complex cellular environments and under various biological conditions including changes in intracellular pH, temperature, and metabolic activity. Furthermore, autofluorescence commonly observed in cells or tissues can be avoided using Qtracker® 655, 705, or 800 Kits.

Long-Lasting, Targeted Signal
Using Qtracker® Cell Labeling Kits, you can observe labeled cells using extensive continuous illumination, without the photobleaching and degradation problems often associated with organic dyes. Qtracker® labels are distributed in vesicles in the cytoplasm, and are inherited by daughter cells for at least six generations. Fluorescence from the Qtracker® labels can be seen up to a week after delivery in some cell lines. Long-term cellular retention makes Qtracker® Cell Labeling Kits ideal for studying cell motility, migration, differentiation, morphology, and many other cellular function studies. Qtracker® labels do not leak out of intact cells to be taken up by adjacent cells in the population.

Monitor the Signal on Multiple Platforms
Qtracker® reagent-labeled live cells can be easily monitored on a variety of platforms, including flow cytometry, fluorescence/confocal microscopy, fluorescence microplate readers, and high-content imaging systems.

Minimal Impact on Live Cells
The cytotoxicity of the materials use in Qtracker® Cell Labeling Kits has been tested in a variety of cell lines including CHO, HeLa, U-118, 3T3, HUVEC, and Jurkat cells. Labeling with Qtracker® Cell Labeling Kits appears to exert minimal impact on cellular surface marker expression, cell proliferation, cellular enzyme activity, and cell motility.

Useful in a Variety of Cell Tracing Studies
Post-labeling, researchers have demonstrated a wide variety of applications for Qtracker® labeled cells, including cell co-culture and cell assembly into heterotypic assemblies, multilineage differentiation, trans-differentiation versus cell fusion, embryonic and mesenchymal stem cell tracking, and cell migration dynamics.

Qtracker™ 655 Vascular Labels (Invitrogen™)

Qtracker® non-targeted quantum dots are designed to be injected into the tail vein of mice for the study of vascular structure using small animal in vivo imaging (SAIVI) techniques. These nanocrystals exhibit intense fluorescence with red-shifted emission for increased tissue penetration, and have a PEG surface coating specially developed to minimize nonspecific interactions and reduce immune response by the tissue. Because the PEG surface coating does not contain reactive functional groups, the Qtracker® non-targeted quantum dots are retained in circulation longer and can be imaged for up to 3 hours with a single injection or for longer periods of time with additional injections.

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

Key Attributes:

Qtracker® 655 label has Ex/Em (405-615/655) nm
Designed for small animal in vivo imaging
Introduced via tail vein injection, can be imaged for up to 3 hours after injection
Available in four colors—565 nm, 655 nm, 705 nm, or 800 nm emission

Read more about SAIVI and about applications for Qdot® nanocrystals.

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

Alexa Fluor™ 555 C2 Maleimide (Invitrogen™)

Alexa Fluor® 555 is a bright orange dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 555 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 555 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® 555 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® 555 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® 555 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 556/572 nm
Extinction coefficient: 158,000 cm-1M-1
Spectrally similar dyes: Tetramethylrhodamine
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™ 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

Alexa Fluor™ 568 Hydrazide, for microinjection (Invitrogen™)

Alexa Fluor® 568 Hydrazide is useful as a cell tracer and as a reactive dye for labeling aldehydes or ketones in polysaccharides or glycoproteins. This version is formatted as a ready-to-use solution that is dissolved in a 200 mM KCl solution and filter sterilized.

Alexa Fluor® 568 is a bright, red fluorescent dye. 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).

Detailed information about this AlexaFluor® hydrazide:

• Fluorophore label : Alexa Fluor® 568 dye
• Reactive group: hydrazide
• Reactivity: Aldehydes or keytones in polysaccharides or glycoproteins
• Ex/Em of the conjugate: 576/599 nm
• Extinction coefficient: 86,000 cm-1M-1
• Spectrally similar dyes: Rhodamine Red
• Molecular weight: 730.74

Cell Tracking and Tracing Applications
Alexa Fluor® hydrazides and hydroxlamines are useful as low molecular weight, membrane-impermeant, aldehyde-fixable cell tracers, exhibiting brighter fluorescence and greater photostability than cell tracers derived from other spectrally similar fluorophores. They are easily loaded into cells by microinjection, infusion from patch pipette, or uptake induced by our Influx™ Pinocytic Cell-Loading Reagent. It is designed to be loaded into cells by microinjection or infusion from patch pipette. Learn more about cell tracking and tracing.

Glycoprotein and Polysaccharide Labeling Applications
The Alexa Fluor® hydrazides and hydroxlamines are reactive molecules that can be used to add a fluorescent label to biomolecules containing aldehydes or ketones. Aldehydes and ketones can be introduced into polysaccharides and glycoproteins by periodate-mediated oxidation of vicinal diols. Galactose oxidase can also be used to oxidize terminal galactose residues of glycoproteins to aldehydes.

Hydrazide vs Hydroxylamine
Hydrazine derivatives react with ketones and aldehydes to yield relatively stable hydrazones. Hydroxylamine derivatives (aminooxy compounds) react with aldehydes and ketones to yield oximes. Oximes are superior to hydrazones with respect to hydrolytic stability. Both hydrazones and oximes can be reduced with sodium borohydride (NaBH4) to further increase the stability of the linkage.

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.

Related Products
DMSO (dimethylsulfoxide) (D12345)
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)

EZ-Link™ Sulfo-NHS-Biotinylation Kit (Thermo Scientific™)

The Thermo Scientific EZ-Link Sulfo-NHS-Biotinylation Kit contains reagents sufficient for 10 biotinylation reactions (e.g., 1–10 mg antibody per reaction). The EZ-Link Sulfo-NHS-Biotin reagent included in the kit is a short-chain, water-soluble biotinylation reagent used for labeling antibodies, proteins and other molecules that have primary amines.

Features of EZ-Link Sulfo-NHS-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
Very short—spacer arm (total length added to target) is 13.5 angstroms; it consists of the native biotin valeric acid group only.

Sulfo-NHS-Biotin is the shortest 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-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-Biotin
EZ-Link™ Micro Sulfo-NHS-Biotinylation Kit

HABA (4'-hydroxyazobenzene-2-carboxylic acid) (Thermo Scientific™)

Thermo Scientific Pierce HABA is 4'-hydroxyazobenzene-2-carboxylic acid, a simple reagent that enables spectrophotometric (colorimetric) estimation of biotinylation levels of labeled proteins and other molecules.

Features of HABA:

• HABA-avidin complex can be used over a wide range of pH and salt concentrations
• Amount of Avidin can be calculated directly from the increased absorbance at 500nm complexing with the HABA Dye
• Calculate results directly from absorbance values based on extinction coefficients using the procedure outlined in the instructions
• Complete kits also available! See Pierce Biotin Quantitation Kit (Part No. 28005) and Fluorescence Biotin Quantitation Kit (Part No. 46610)

Determine the molar ratio of biotin incorporated into a protein using the HABA-Avidin method. HABA dye (4'-hydroxyazobenzene-2-carboxylic acid ) binds to avidin to produce a yellow-orange colored complex which absorbs at 500nm. Free biotin will displace the HABA dye and cause the absorbance to decrease. A standard curve can be established using the free biotin to estimate the number of moles of biotin incorporated after biotinylating a protein. View online HABA Calculator.

pHrodo™ Red, succinimidyl ester (pHrodo™ Red, SE) (Invitrogen™)

New pH-sensitive pHrodo™ Red dye conjugates give faster and more accurate results than any other phagocytosis assay

pHrodo™ Red dye conjugates are non-fluorescent outside the cell, but fluoresce brightly red in phagosomes

Get faster staining and more accurate results - without the need for wash steps or quencher dye


• Specific detection of phagocytosis and endocytosis
• Reduced signal variability and improved timing in sensitive experiments
• Multiplex with green dyes such as GFP, Fluo-4, or calcein


The fluorescence of the novel pHrodo™ Red dye dramatically increases as pH decreases from neutral to the acidic, making it an ideal tool to study phagocytosis and its regulation by drugs and/or environmental factors. The lack of fluorescence outside the cell eliminates the need for wash steps and quencher dyes.

Use the ready-made pHrodo™ Red E.coli BioParticles® conjugates in imaging or flow applications, or pHrodo™ Red SE, the activated succinimidyl ester, for labeling microorganisms or proteins of your choice.

BODIPY™ 581/591 NHS Ester (Succinimidyl Ester) (Invitrogen™)

BODIPY® 581/591 dye is bright, red fluorescent dye with similar excitation and emission to Rhodamine Red™ and Alexa Fluor®568. It has a high extinction coefficient and fluorescence quantum yield and is relatively insensitive to solvent polarity and pH change. In contrast to the highly water soluble fluorophores Alexa Fluor® 488 dye and fluorescein (FITC), BODIPY® dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds. BODIPY® 581/591 dye has a relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization-based assays and a large two-photon cross-section for multiphoton excitation. In addition to reactive dye formulations, we offer BODIPY® 581/591 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 BODIPY® 581/591 is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY® 581/591 conjugates exhibit bright fluorescence, narrow emission bandwidths, and relatively long excited-state lifetimes, which can be useful for fluorescence polarization assays and two-photon excitation (TPE) microscopy.

This reactive dye contains a C3 alkyl spacer between the fluorophore and the NHS 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.

Detailed information about this BODIPY® 581/591 NHS ester:

Fluorophore label: BODIPY® 581/591 dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 581/591 nm
Extinction coefficient: 136,000 cm-1M-1
Molecular weight: 489.28

Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. The reaction can be scaled 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 BODIPY® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), 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 BODIPY® 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 medium 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™ 532 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 532 is a bright yellow 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 NHS ester (or succinimidyl ester) of Alexa Fluor® 532 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® 532 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 530/555 nm
Extinction coefficient: 81,000 cm-1M-1
Molecular weight: 723.8

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.

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

Thermo Scientific EZ-Link Amine-PEG2-Biotin is a water-soluble biotin compounds containing a polyethylene glycol (PEG) spacer arm and a terminal primary amine for conjugation via EDC and other crosslinker methods.

Features of EZ-Link Amine-PEG2-Biotin:

Biotinylation—label molecules and surfaces for assays or affinity purification methods involving avidin or streptavidin probes and resins
Amine-activated—primary amine can be crosslinked to proteins and material surfaces using EDC and other crosslinkers
Pegylated—polyethylene glycol (PEG) groups in spacer arm enhances water solubility of biotinylated molecules
Medium length—spacer arm (total length added to target) is 20.4 angstroms

Amine-PEG2-Biotin and Amine-PEG3-Biotin are the shorter two of three amine-modified biotin compounds that contain polyethylene glycol (PEG) spacer arms. The short PEG segments are hydrophilic and confer greater solubility to labeled proteins compared to reagents having only hydrocarbon spacers. The primary amines of these pegylated biotin reagents can be conjugated to carboxyl groups on carboxy termini, aspartate residues or glutamate residues using EDC (Part No. 22980), a water-soluble carbodiimide crosslinker. EDC activates carboxyl groups to bind to the—NH2 group of the amino-biotin, forming an amide bond.

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

Amino-biotin compounds can be conjugated to functional groups of proteins and other molecules in a variety of ways. The most common method is to crosslink the terminal primary amine to carboxyl groups using . Carboxyl groups (-COOH) occur in aspartate or glutamate residues and the carboxy-terminus of polypeptides. When activated with EDC (Part No. 22980), carboxylates react with amino (—NH2) groups to form amide bonds. Carboxylate molecules and surface materials can be pre-activated using EDC with Sulfo-NHS (Part No. 24510) for subsequent reaction to primary amines (see NHS-ester Chemistry).

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Click-iT™ SDP Ester sDIBO Alkyne (Invitrogen™)

Click-iT SDP Ester sDIBO Alkyne reacts with azides via a copper-free Click chemistry reaction to produce SDP Ester (amine-reactive) bioconjugates. sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less “sticky” and give lower signal background in biological samples. Copper-free Click bio-conjugation reactions are ideal for surface labeling of live cells and also minimize damage to enzymes and fluorescent proteins like GFP or R-PE. Macromolecules that have been azide-modified enzymatically, chemically, or metabolically can be now be labeled easily, yielding more soluble bioconjugates with improved biological labeling utility.

• More soluble than DIBO cyclooctynes leading to more soluble conjugates
• Minimal background potential in cells and tissues compared to original DIBO cycloctynes

R-Phycoerythrin, Pyridyldisulfide Derivative (Invitrogen™)

The pyridyldisulfide derivative of R-phycoerythrin (R-PE) can be reacted directly with thiolated antibodies, enzymes and other biomolecules to form a disulfide linkage.

pHrodo™ Green STP Ester (Invitrogen™)

The amine-reactive pH-sensitive pHrodo® Green STP ester 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 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 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.

The pHrodo® Green STP ester is an amine reactive dye that can be used to make pHrodo® Green 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.

Amine reactive pHrodo® dye is also available with red fluorescence (see pHrodo® Red SE). In addition, thiol-reactive pHrodo® Red Maleimide and Green Maleimide are a great alternative to amine-reactive dyes for antibody labeling. Thiol labeling ensures that the dye will not attach to the binding region of the antibody. pHrodo® Green and Red conjugates, for example dextran, E. coli and S. aureus, are also available in ready-to-use form.

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

CellVue™ Jade Cell Labeling Kit (Invitrogen™)

CellVue™ dyes are lipophilic dyes that can be used to label the cell membrane for the purpose of identifying and tracking labeled cells. Cell labeling is rapid and stable and can be combined with fluorescently labeled antibodies and other markers of cellular function for flow cytometric analysis and fluorescent microscopy. Mini CellVue™ Kits are supplied with one vial of dye stock (1 mM in ethanol) and one vial of labeling vehicle (Diluent C).

CellVue™ Jade is a green fluorescent cell labeling reagent. It is optimally excited at 478 nm and has a peak emission of 508 nm. CellVue™ Jade is compatible with most multi-color applications; however, due to similar emission properties it cannot be used in combination with FITC or Alexa Fluor™ 488 reagents.

Reported Application
Flow Cytometric Analysis, Microscopy, Immunocytochemistry, Cell Labeling

DyLight™ 775-B3 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight near-infrared specialty dyes, comparable to Alexa Fluor and IRDye NIR dyes, can be used to label antibodies, peptides, and other proteins at primary amines. DyLight 775-B3 dye has a structure based on the benzopyrillium core, with 3 sulfonates. It has excitation and emission peaks at 770 and 788 nm, respectively (in ethanol).

General characteristics of DyLight near-infrared emitting specialty dyes:

Large selection—the largest family of dyes available for NIR fluorescence applications
NHS ester reactive group—allows immediate labeling of antibodies, proteins, peptides and other amine-containing molecules through amide bond formation
Broad spectrum of water solubilities—choose from hydrophilic to hydrophobic dyes to optimize the right dye label for the best performance in a given application
NIR dyes avoid background interference—DyLight NIR Dyes avoid fluorescence interference or quenching effects from biomolecules present in samples
Excellent signal penetration through cells and tissues—DyLight NIR Dyes provide the optimal window for excitation and emission for in vivo imaging applications

DyLight NIR Dyes are a family of labeling agents that can be used for bright fluorescence detection in cell-based imaging or in vivo imaging applications. NIR 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. NIR dyes are best for imaging through tissues and away from indigenous fluorescent biomolecule interference or quenching. DyLight Near Infrared Dyes represent the largest selection of fluorescent labels that are commercially available.

Criteria to consider when choosing a DyLight NIR Specialty Dye
• Excitation and emission wavelengths—choose the best dye to match the excitation and emission capabilities of your instrument
• Water solubility—choose a DyLight NIR 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 NIR dyes allows a number of candidate dyes to be tested in a given application for optimal performance.

Applications:
In vivo or ex vivo imaging
• Tumor imaging with labeled peptides
• NIR fluorescence (NIRF) imaging of labeled silica nanoparticles
• NIR in vitro imaging and characterization
• Determination of thermal stability
• Cytotoxicity assays
• Molecular imaging
• UV-VIS-NIR spectroscopy
• Fluorescence correlation spectroscopy
• MRI applications
• DNA sequencing
• Primer labeling for PCR
• 2-D gel electrophoresis
• Flow cytometry/fluorescence-activated cell sorting (FACS)
• Laser scanning confocal microscopy

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DyLight™ 775-B4 NHS Ester

DyLight™ 800 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 800 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 800 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

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. DyLight 800 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of DyLight 800 NHS Ester:

High performance— DyLight 800 replaces Alexa Fluor 800 and IRDye 800 for near-infrared staining
Specific— NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Optimized procedure— following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications:
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 800 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 800 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

We also offer Standard and Microscale DyLight 800 Antibody Labeling Kits for fast and efficient fluorescent labeling of antibodies for use in fluorescence methods. The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG. Both kit sizes include the Amine-Reactive DyLight 800 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

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DyLight™ 800 Antibody Labeling Kit
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DyLight™ 350 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 350 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 350 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

DyLight 350 is a derivative of aminomethylcoumarine acetate (AMCA) and has a higher fluorescence intensity than Alexa Fluor™ 350 and AMCA 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. DyLight 350 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of DyLight 350 NHS Ester:

High performance—DyLight 350 shows brighter fluorescence than Alexa Fluor 350 and AMCA
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 350 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require DyLight 350 Amine-Reactive Dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 350 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

We also offer Standard and Microscale DyLight 350 Antibody Labeling Kits for fast and efficient fluorescent labeling of antibodies for use in fluorescence methods.The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG. Both kit sizes include the Amine-Reactive DyLight 350 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

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R-Phycoerythrin (Thermo Scientific™)

Thermo Scientific R-Phycoerythrin is a red-fluorescent, multi-subunit protein that can be crosslinked or conjugated to other molecules to make fluorescent probes.

Features of R-Phycoerythrin:

R-Phycoerythrin—red-fluorescent, multi-subunit protein in the phycobiliprotein family
Large Stokes shift —excitation (absorption) at one of three wavelengths produces light emission at a much higher wavelength

This fluorochrome is a member of the phycobiliprotein family of proteins isolated from the marine algae Porphyra tenera or Gastroclonium coulterii. Fluorochrome-labeled reagents have high resolution and are therefore advantageous in immunological assays, such as live cell staining, double-labeling techniques and cell sorting procedures. The strong absorption bands of the R-Phycoerythrin are in the visible region of the spectrum, extending from the green to the far-red wavelengths. The absorbance spectra extends over a broad range of potential excitation wavelengths, allowing for versatility in the excitation source and creating large Stokes shifts, thus minimizing interference from Rayleigh-scattered light.

Properties of R-Phycoerythrin:

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