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Qdot™ 565 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 565 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® 565 ITK™ carboxyl quantum dot has emission maxima of ~565 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.

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.

EZ-Link™ Phosphine-PEG3-Biotin (Thermo Scientific™)

Thermo Scientific Pierce EZ-Link Phosphine-PEG3-Biotin is a biotinylation reagent for labeling azide-containing molecules, which enables biotin-based detection and affinity purification of molecules via Staudinger ligation strategies.

Features of EZ-Link Phosphine-PEG3-Biotin:

Soluble—easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
Compatible—reaction chemistry occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
Chemoselective—the phosphine reactive group is specific in biological samples for bioorthogonal azide-tagged molecules, ensuring that biotinylation is specific
PEG spacer—polyethylene glycol spacer arm helps maintain solubility of labeled molecules and decreases steric hindrance for affinity-binding to avidin, streptavidin or NeutrAvidin Protein

When used in combination with azide labeling strategies, this compound enables detection or affinity purification of protein interactions and post-translational modifications using streptavidin probes or streptavidin agarose resins. The phosphine group of Phosphine-PEG3-Biotin conjugates to azide groups by the Staudinger reaction mechanism. Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-tagged derivatives of naturally occurring metabolic building blocks. Because neither phosphines nor azides are present in biological systems, they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.

Pierce™ NHS-Rhodamine Antibody Labeling Kit (Thermo Scientific™)

Thermo Scientific NHS-Rhodamine is a high-performance derivative of rhodamine dye, activated for easy and reliable labeling of antibodies, proteins and other molecules for use as fluorescent probes. Use our NHS-Rhodamine Antibody Labeling Kit to label and purify antibodies in about one hour.

Features of NHS-Rhodamine :

Amine-specific labeling—NHS-ester varieties of rhodamine efficiently label antibodies and other purified proteins at primary amines (lysine side chains)
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios for optimum activity and fluorescence
Single-use fluors—no need to weigh tiny amounts powder; kit contains single-use vials of reagent
Efficient purification—kit includes purification resin and easy-to-use spin columns, ensuring rapid and efficient removal of non-reacted dye and excellent protein recovery

NHS Rhodamine is an amine-reactive derivative of rhodamine dye that has wide-ranging application as antibody and other probe labels for use in fluorescence microscopy, flow cytometry and immunofluorescence-based assays such as western blotting and ELISA.

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

Properties of Rhodamine Dyes:
Thermo Scientific Pierce Rhodamine Dyes are mixtures of isomers with reactive groups attached at the 5- and 6-positions of the bottom ring. The properties of these isomers are indistinguishable in terms of excitation and emission spectra, and for protein applications there is no need to isolate a specific isomer.

NHS-Rhodamine is activated with the N-hydroxy-succinimidyl-ester (NHS ester) functional group. Compared to TRITC, the NHS-ester deriviative has greater specificity toward primary amines in the presence of other nucleophiles and results in a more stable linkage following labeling.

Application Data:

Related Products
NHS-Rhodamine (5/6-carboxy-tetramethyl-rhodamine succinimidyl ester), mixed isomer
TRITC (5/6-tetramethyl-rhodamine isothiocyanate), mixed isomer

APEX™ Alexa Fluor™ 488 Antibody Labeling Kit (Invitrogen™)

The APEX® Antibody Labeling Kits are our best option for covalently attaching a fluorophore to small amounts of IgG antibody (~10–20 μg). It is ideal for the efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Labeled antibodies are ready for use in imaging or flow cytometry applications in as little as 2.5 hours with very little hands on time.

Important Features of Alexa Fluor® APEX® Antibody Labeling Kits:

• Labeled antibodies typically ready to use in 2.5 hours (~15 minutes hands on time)
• Designed to label 10–20 μg of IgG
• Covalent attachment
• Compatible with contaminating proteins or stabilizers like BSA
• No columns needed; everything you need is supplied for 5 separate labelings
• Choose from Alexa Fluor® 488, 555, 568, 594, and 647 dyes, Oregon Green® 488 dye, Pacific Blue™ dye, and Biotin-XX.


Better Results and Workflows With Primary Labeled Antibodies
A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding. Further, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment if they are directly labeled with compatible fluorophores.

Contaminating Proteins or Protein Stabilizers Are Not a Problem
Many IgG antibodies are often available only in small quantities and packaged with stabilizing proteins, such as BSA, or other contaminants which can interfere with the amine-reactive labeling reagents. The APEX® Antibody Labeling Kits avoids this by utilizing a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX® antibody labeling tip. Contaminants are simply eluted through the tip, prior to applying the amine-reactive label.

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.

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

Thermo Scientific EZ-Link NHS-SS-Biotin enables simple and efficient biotinylation of antibodies, proteins and other primary amine-containing molecules, as well as intracellular labeling.

Features of EZ-Link NHS-SS-Biotin:

Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
Membrane-permeable—can be used to label inside cells (intracellular)
Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
Cleavable—disulfide bond in spacer arm allows the biotin label to be removed using reducing agents such as DTT; only a small sulfhydryl group remains attached to the molecule
Solubility—must be dissolved in DMSO or DMF before further dilution in aqueous buffers
Medium length—spacer arm (total length added to target) is 24.3 angstroms; it consists of the native biotin valeric acid group extended by a 7-atom chain

NHS-SS-Biotin is succinimidyl 2-(biotinamido)-ethyl-1,3' -dithiopropionate, a compound that enables simple and efficient biotinylation of antibodies, proteins and other primary amine-containing molecules. The extended spacer arm (24.3 angstroms) of this reagent reduces steric hindrance associated with binding to avidin or other biotin-binding proteins and incorporates a reducing agent-cleavable disulfide bond (-S-S-) within the spacer, providing further versatility to the reagent.

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.

DyLight™ 594 Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 594 Antibody Labeling Kit contains 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. 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.

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 available as a stand-alone reagent or 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)
Convenient kit sizes—standard and microscale sizes are offered to match your experimental needs
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.

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

Alexa Fluor™ 488 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.

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

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

Alexa Fluor™ 680 Antibody Labeling Kit (Invitrogen™)

Molecular Probes® Alexa Fluor® antibody labeling kits provide a convenient means to label small amounts of antibodies with Alexa Fluor® dyes (choice of 10 colors). This kit is optimized for labeling 100 μg of antibody per reaction with infrared fluorescent Alexa Fluor® 680. Small amounts of other proteins (>40 kDa) can also be labeled with this kit.

The Alexa Fluor® 680 Antibody Labeling Kits contains everything you need to perform five separate labeling reactions as well as to purify the resulting conjugates. The conjugates are ideal for multiple applications, including small animal in vivo imaging, flow cytometry, fluorescent microscopy, immunohistochemistry, primary detection, ELISAs, immunocytochemistry, indirect FISH, and more.

With an excitation and emission maximum of 684/707 nm, Alexa Fluor® 680 can be efficiently excited and detected in most standard small animal in vivo imaging and fluorescent western blot imaging systems.

Important Features of Alexa Fluor® 680 Antibody Labeling Kit:

• Labeled proteins typically ready to use in 90 min (~15 min hands-on time)
• Optimized for small-scale labeling of any protein >40 kDa
• Conjugates purified using convenient spin filters
• Stabilizing proteins must be removed from the sample before labeling
• Includes detailed instructions for determining degree of labeling (DOL)

Better Results and Workflows Possible with Labeled Primary Antibodies
A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding. Further, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment if they are directly labeled with compatible fluorophores.

Superior 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.

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.

APEX™ Alexa Fluor™ 594 Antibody Labeling Kit (Invitrogen™)

The APEX® Antibody Labeling Kits are our best option for covalently attaching a fluorophore to small amounts of IgG antibody (~10–20 μg). It is ideal for the efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Labeled antibodies are ready for use in imaging or flow cytometry applications in as little as 2.5 hours with very little hands on time.

Important Features of Alexa Fluor® APEX® Antibody Labeling Kits:

• Labeled antibodies typically ready to use in 2.5 hours (~15 minutes hands on time)
• Designed to label 10–20 μg of IgG
• Covalent attachment
• Compatible with contaminating proteins or stabilizers like BSA
• No columns needed; everything you need is supplied for 5 separate labelings
• Choose from Alexa Fluor® 488, 555, 568, 594, and 647 dyes, Oregon Green® 488 dye, Pacific Blue™ dye, and Biotin-XX.


Better Results and Workflows With Primary Labeled Antibodies
A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding. Further, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment if they are directly labeled with compatible fluorophores.

Contaminating Proteins or Protein Stabilizers Are Not a Problem
Many IgG antibodies are often available only in small quantities and packaged with stabilizing proteins, such as BSA, or other contaminants which can interfere with the amine-reactive labeling reagents. The APEX® Antibody Labeling Kits avoids this by utilizing a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX® antibody labeling tip. Contaminants are simply eluted through the tip, prior to applying the amine-reactive label.

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.

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

CPM (7-Diethylamino-3-(4'-Maleimidylphenyl)-4-Methylcoumarin) (Invitrogen™)

The thiol-reactive coumarin, CPM is very weakly fluorescent until reacted with thiols producing a conjugate with excitation/emission maxima of ~384/470 nm.

DyLight™ 755 NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 755 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 755 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

DyLight 755 is a near-IR fluor that is invisible to the naked eye but increases the staining options when using infrared imaging systems. DyLight 755 has spectral properties that are very similar to other near-IR dyes, including Alexa Fluor™ 750. 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 755 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of DyLight 755 NHS Ester:

High performance—DyLight 755 replaces Alexa Fluor 755 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 755 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 755 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 755 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 755 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™ 755 Antibody Labeling Kit
DyLight™ 755 Microscale Antibody Labeling Kit

Pierce™ Streptavidin, Hydrazide-Activated (Thermo Scientific™)

Thermo Scientific Pierce Hydrazide-Activated Streptavidin conjugate include recombinant streptavidin in a purified form activated for crosslinking to carbonyl groups in a molecule.

Related Products
Pierce™ Streptavidin
Pierce™ Streptavidin, Horseradish Peroxidase Conjugated
Pierce™ Streptavidin, Alkaline Phosphatase Conjugated
Pierce™ Streptavidin, Maleimide-Activated

CellTrace™ Calcein Green, AM - Special Packaging (Invitrogen™)

CellTrace calcein green AM is a cell-permeant dye that can be used to determine cell viability in most eukaryotic cells. In live cells the nonfluorescent CellTrace calcein green AM is converted to a green-fluorescent calcein after intracellular esterases remove the acetoxymethyl (AM) esters. This dye is also available as 1 mg of the solid (C-1430) and resuspended in DMSO (C-3099). For a longer wavelength version of this dye, check out our CellTrace calcein red-orange AM (C-34851).

Zenon™ Alexa Fluor™ 488 Mouse IgG1 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

CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ CFSE Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Non-toxic—staining does not adversely effect cell health
• Simple, robust staining protocol

View a selection guide for all CellTrace™ Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ CFSE dye enables the visualization of eight or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace™ CFSE dye easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye is designed to provide a consistent signal, even after several days in a cell culture environment.

Non-toxic
CellTrace™ CFSE dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells. Researchers have used CFSE SE labeling to show that transplantable hematopoietic cells proliferate in vitro in response to stimulation by a growth factor cocktail.

Simple, robust staining protocol
The CellTrace™ CFSE Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

Qtracker™ 655 Cell Labeling Kit (Trial Size) (Invitrogen™)

The Qtracker® 655 Cell Labeling Kit uses a custom targeting peptide to deliver far red-fluorescent Qdot® 655 probe (ex/em 405–615/655 nm) into the cytoplasm of live cells. Once inside the cells, the Qdot® 655 probe provides an intense, stable fluorescence that can be traced through several generations and is not transferred to adjacent cells in a population. This trial-size version offers a convenient introduction to the Qtracker® cell labeling technology at an affordable price.

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

Features of the Qtracker® Cell Labeling kits include:

• Qtracker® probes allow for continuous illumination, without photobleaching or degradation problems often associated with organic dyes
• Simple to use—add Qtracker® labeling solution to cells in serum-containing media, incubate one hour, then detect and track the cells
• Provide an intense, stable fluorescence that can be traced through several generations
• Excellent tool 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® probes into the cytoplasm of live cells. Since the cytoplasmic delivery mechanism is not mediated by a specific enzyme, no cell-type specificity has been observed. Maximum delivery is typically accomplished in less than 1 hour.

The Qdot® probes are compatible with serum-sensitive cells. The intense fluorescence from the Qdot® probes is maintained in complex cellular environments and under various biological conditions including changes in intracellular pH, temperature, and metabolic activity.

Long-Lasting, Targeted Signal
Use of Qtracker® Cell Labeling kits results in the ability to observe labeled cells with extensive continuous illumination, without photobleaching or degradation problems often associated with organic dyes. Once inside the cells, the Qdot® probes are localized to cytoplasmic vesicles and are inherited by daughter cells for at least six generations. Fluorescence from the Qdot® probes can be detected for up to a week after delivery in some cell lines. The long-term cellular retention of the Qdot® probes results in an ideal tool for studying cell motility, migration, differentiation, morphology, and many other cellular function studies. Since the Qtracker® labels do not leak out of intact labeled cells and are not transferred to adjacent cells, the Qtracker® Cell Labeling kits result in a targeted signal.

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 used 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.

ATTO-TAG™ FQ Derivatization Reagent (FQ; 3-(2-Furoyl)quinoline-2-Carboxaldehyde) (Invitrogen™)

The ATTO-TAG FQ reagent allows the ultrasensitive detection of primary amines. This reagent is available as a standalone product or as a component of an amine-derivatization kit (A-2334). Because ATTO-TAG FQ is nonfluorescent in the unreacted state and reacts specifically with amines to form highly fluorescent conjugates, it is particularly well-suited to the derivatization of peptides, amino acids, aminated sugars and other amine-containing molecules for detection by capillary electrophoresis with laser-induced fluorescence (LIF). Recently, researchers described a simple method in which the ATTO-TAG FQ reagent was used to derivatize very dilute solutions (10-8 M) of peptides.

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

The Thermo Scientific EZ-Link Sulfo NHS-SS Biotinylation Kit contains sufficient reagents for 10 biotinylation reactions (e.g., 1–10 mg antibody per reaction). The EZ-Link Sulfo-NHS-SS-Biotin included in the kit is a water-soluble, NHS-ester biotinylation reagent whose spacer arm includes a cleavable disulfide bond for reversible labeling of proteins and cell surface primary amines.

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

Protein labeling – biotinylate antibodies to facilitate immobilization, purification or detection
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
Cleavable—disulfide bond in spacer arm allows the biotin label to be removed using reducing agents such as DTT; only a small sulfhydryl group remains attached to the molecule
Soluble – charged sulfo-NHS group increases reagent water solubility compared to ordinary NHS-ester compounds
Medium length—spacer arm (total length added to target) is 24.3 angstroms; it consists of the native biotin valeric acid group extended by a 7-atom chain

Sulfo-NHS-SS-Biotin is a thiol-cleavable amine-reactive biotinylation reagent that contains an extended spacer arm to reduce steric hindrances associated with avidin binding. This reagent is water-soluble, enabling biotinylation to be performed in the absence of organic solvents such as DMSO or DMF for applications that cannot tolerate solvents or are complicated by their inclusion. The compound is particularly useful for labeling and purifying cell surface proteins, because (a) its sulfonate group prevents it from permeating cell membranes and (b) its cleavable spacer arm enables initially biotinylated proteins to be released from streptavidin affinity columns.

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-SS-Biotin
EZ-Link™ Micro Sulfo-NHS-SS-Biotinylation Kit

Alexa Fluor™ 488 Alkyne (Alexa Fluor™ 488 5-Carboxamido-(Propargyl), Bis(Triethylammonium Salt)), 5-isomer (Invitrogen™)

The green-fluorescent Alexa Fluor® 488 alkyne is reactive with azides via a copper-catalyzed click reaction. In addition to being an exceedingly bright and photostable fluorophore for use with flow cytometry, microscopy and HCS, Alexa Fluor® 488 can also be utilized as a bio-orthogonal or biologically unique hapten for use in applications requiring signal amplification.

Pierce™ NHS-Fluorescein Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific NHS-Fluorescein Antibody Labeling Kit is a multi-component kit containing a high-performance derivative of fluorescein dye, activated for easy and reliable labeling of antibodies, proteins and other molecules for use as fluorescent probes.

Features of the kit:

Easy —convenient kit with NHS-fluorescein to label and purify antibody in about one hour
Amine-specific labeling—the isothiocyanate variety of fluorescein efficiently labels antibodies and other purified proteins at primary amines (lysine side chains)
Optimized kit procedure—following the standard protocol results in antibodies with excellent dye:protein ratios for optimum activity and fluorescence
Single-use fluors—no need to weigh tiny amounts of powder; kits contain single-use vials of reagent
Efficient purification—kits include purification resin and easy-to-use spin columns, ensuring rapid and efficient removal of non-reacted dye and excellent protein recovery

NHS-fluorescein is activated with the N-hydroxy-succinimidyl-ester (NHS ester) functional group. Compared to FITC, the NHS-ester deriviative has greater specificity toward primary amines in the presence of other nucleophiles and results in a more stable linkage following labeling. Pierce Amine-reactive Fluorescein Dyes are mixtures of isomers with reactive groups attached at the 5- and 6-positions of the bottom ring. The properties of these isomers are indistinguishable in terms of excitation and emission spectra, and for protein applications there is no need to isolate a specific isomer.

Properties of NHS-Fluorescein:

• Alternative name: 5/6-FAM SE
• Chemical name: 5/6-carboxyfluorescein succinimidyl ester
• Molecular weight: 473.4
• Excitation source: 488 nm spectral line, argon-ion laser
• Excitation wavelength: 494 nm
• Emission wavelength: 518 nm
• Extinction coefficient: > 70,000 M-1cm-1
• CAS #: 117548-22-8
• Purity: > 90% by HPLC
• Solubility: Soluble in DMF or DMSO
• Reactive groups: NHS ester, reacts with primary amines at pH 7.0 to 9.0

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

Related Products
NHS-Fluorescein (5/6-carboxyfluorescein succinimidyl ester), mixed isomer
FITC (5/6-fluorescein isothiocyanate), mixed isomer
Pierce™ FITC Antibody Labeling Kit

FluoReporter™ Biotin Quantitation Assay Kit, for biotinylated proteins (Invitrogen™)

The FluoReporter® Biotin Quantitation Assay Kit provides a sensitive fluorometric assay for determining the number of biotin labels on nucleic acid samples (i.e., cDNA). The assay uses Biotective™ Green reagent, which contains a fluorescent dye that is quenched by ligands occupying the biotin binding sites. In the presence of biotin, the quencher dye ligands are displaced and fluorescence occurs. The fluorescence is proportional to the amount of added biotin. The results can be read with any fluorescence-based microplate reader capable of detecting FITC dye.

Important Features of FluoReporter® Biotin Quantitation Assay Kits:
• Can detect from 4 to 80 picomoles of biotin in a sample (50-fold higher sensitivity than HABA biotin-binding assay)
• Can be applied to as little as 13 ng of biotin-labeled nucleic acid (more is needed for lower degrees of labeling)
• Excitation/emission maxima of the reagent is 495/519 nm
• Supplied with a biotinylated positive control for standard curve


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

Fluorescein Cadaverine; 5-((5-Aminopentyl)thioureidyl)Fluorescein, Dihydrobromide Salt (Invitrogen™)

The primary aliphatic amine of fluorescein cadaverine 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™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit (Invitrogen™)

The Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit provides a fast, sensitive, non-toxic, and non-radioactive method for the detection of nascent protein synthesis utilizing fluorescence microscopy, high-content imaging, or flow cytometry. Included in the kit are L-homopropargylglycine (HPG), an amino acid analog of methionine containing an alkyne moiety, and Alexa Fluor® 488 azide. The HPG is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of the Alexa Fluor® 488 azide leads to a chemoselective ligation or "click" reaction between the green fluorescent azide and the alkyne, allowing the modified proteins to be detected by imaged-based analysis.

Non-radioactive alternative—an alternative to the traditional 35S-methionine
Visualize bulk protein dynamics—fluorescent tagging of proteins allows their localization to be determined, including aggregation
Specificity—selective, specific reaction between label and detection tags
Stability —product contains an irreversible, covalent bond
Multiplex-enabled—use in conjuction with Click®-iT AHA (azide amino acid and alkyne dye) to detect spatial and temporal differences
Applicability to biological samples—easy detection; high sensitivity and low background, regardless of complexity

The Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit has been successfully tested in HeLa, A549, and U-2 OS cells with a variety of reagents that inhibit protein synthesis, including cycloheximide and anisomycin. The applicability of these probes to monitor protein degradation has also been shown using inhibitors of the proteasome (MG132 and Bortezomib) and blockers of autophagy (chloroquine) in HeLa cells.

Additionally, due to differences in Click-iT® chemistry between the Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit and Click®-iT AHA with Alexa Fluor® 594 Alkyne, these reagents can be used in conjunction for spatial or temporal determination of differences in nascent protein synthesis.

Alexa Fluor™ 647 Antibody Labeling Kit (Invitrogen™)

Molecular Probes® Alexa Fluor® Antibody Labeling Kits provide a convenient means to label small amounts of antibodies with Alexa Fluor® dyes (choice of 10 colors). This kit is optimized for labeling 100 µg of antibody per reaction with far red fluorescent Alexa Fluor® 647. Comparably small amounts of other proteins (>40 kDa) can also be labeled.

The kit contains everything you need to perform five separate labeling reactions as well as to purify the resulting conjugates. Conjugates are ideal for multiple applications, including flow cytometry, fluorescent microscopy, immunohistochemistry, primary detection, ELISAs, immunocytochemistry, indirect FISH, and more.

Important Features of Alexa Fluor® 647 Antibody Labeling Kit:
• With an excitation and emission maximum of 650/668 nm, Alexa Fluor® 647 can be efficiently excited using a 633 or 635 nm Kr or He-Ne laser line and detected under standard APC/Cy5® filters
• Labeled proteins typically ready to use typically in 90 min (~15 min hands-on time)
• Useful for labeling 100 µg of protein
• Optimized for small-scale labeling of any protein >40 kDa
• Purified using convenient spin filters
• Stabilizing proteins must be removed from the sample before labeling
• Includes detailed instructions for determining degree of labeling (DOL)


Better Results and Workflows with Primary labeled antibodies
A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding. Further, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment if they are directly labeled with compatible fluorophores.

Superior 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 Zor 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 for use in diagnostic procedures.

Alexa Fluor™ 594 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 594 is a bright red dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 594 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 594 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The NHS ester (or succinimidyl ester) of Alexa Fluor® 594 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® 594 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 590/617 nm
Extinction coefficient: 92,000 cm-1M-1
Spectrally similar dyes: Texas Red®, Bodipy-TR
Molecular weight: 819.8

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.

CellVue™ Burgundy 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. The Mini CellVue™ Kits are supplied with one vial of dye stock (1 mM in ethanol) and one vial of labeling vehicle (Diluent C).

CellVue™ Burgundy is a far-red/near-infrared fluorescent cell labeling reagent. It is optimally excited at 683 nm and has a peak emission of 707 nm. CellVue™ Burgundy can be excited by the red laser line (633 nm), however, it can be detected equally in filter sets designed for Alexa Fluor™ 700 and APC-eFluor™ 780 or similar fluorochromes. Therefore, it is not recommended to use antibodies conjugated to these fluorochromes when using CellVue™ Burgundy. CellVue™ Burgundy can be used in combination with APC.

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

7-Diethylaminocoumarin-3-Carboxylic Acid, Succinimidyl Ester (Invitrogen™)

The amine-reactive coumarin, 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester 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).

Azido, PEO (4 propionic Acid, Succinimidyl Ester (3-(Azidotetra(Ethyleneoxy))Propionic Acid, Succinimidyl Ester)) (Invitrogen™)

Conjugates prepared with the amine-reactive alkyne, succinimidyl ester can be detected with an azide-containing molecule in a click chemistry reaction. Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Click reactions have several characteristics: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; the components of the reaction are bioinert; and perhaps most importantly, no side reactions occur – the label and detection tags react selectively and specifically with one another. Unlike traditional chemical reactions utilizing succinimidyl esters or maleimides that target amines and sulfhydryls – functional groups that are not unique – click chemistry-labeled molecules can be applied to complex biological samples and be detected with unprecedented sensitivity due to extremely low background.

QSY™ 9 C5-Maleimide (Invitrogen™)

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

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.

Click-iT™ Alexa Fluor™ 555 sDIBO Alkyne (Invitrogen™)

Click-iT Alexa Fluor 555 sDIBO Alkyne reacts with azides via a copper-free Click chemistry reaction to produce fluorescent Alexa Fluor 555 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

Learn more about Alexa Fluor dyes ›

pHrodo™ Red Maleimide (Invitrogen™)

The thiol-reactive pH-sensitive pHrodo® Red Maleimide dye is suitable for the creation of bioconjugates to study endocytosis and phagocytosis. pHrodo® Red dramatically increases fluorescence as the pH of its surroundings become more acidic.
• Use pH-sensitive pHrodo® Red Maleimide to make pH-sensitive bioconjugates of your choice
• Get faster, more accurate results than with any other endocytosis or phagocytosis assay—no need for wash steps or quenchers
• Multiplex with green fluorescent markers such as GFP, pHrodo® Green, and many others

The increase in fluorescence of pHrodo® Red as pH changes from basic to acidic correlates with the acidification of intracellular vesicles, making it an ideal tool to study endocytosis or phagocytosis and their regulation by environmental factors, drugs, or pathogens. The spectral properties of pHrodo® Red makes it useful for multi-color experiments. pHrodo® Red has been validated for use on a variety of platforms including flow cytometry, fluorescent microscopy, and high content screening (HCS). The lack of fluorescence of pHrodo® Red in a typical extracellular environment eliminates the need for wash steps or quencher dyes in the experimental workflow.

pHrodo® Red Maleimide is a thiol-reactive dye that can be used to create pHrodo® Red bioconjugates in aqueous buffer. The maleimide reacts with free sulphydryl groups produced by the reduction of cysteines in proteins or peptides. Maleimides are particularly useful for labeling antibodies as the dye will not attach to the antibody binding site. This reaction will result in a stable conjugate that can be used in live cell assays or stored for later use.

pHrodo® Red is also available in an amine-reactive form (see pHrodo® Red SE), as well as a selection of ready-to-use conjugates (e.g., E. coli, S. aureus, and dextran). In addition, pHrodo® Green reactive dyes and ready-to-use conjugates are available as a color alternative with the same properties.

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

EZ-Link™ BMCC-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link BMCC-Biotin is a maleimide-activated, sulfhydryl-reactive biotinylation reagent with an extended spacer arm that contains a stabilizing cyclohexane group.

Features of EZ-Link BMCC-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
Membrane-permeable—can be used to label inside cells (intracellular)
Thiol-reactive—reacts with sulfhydryls (-SH), such as the side-chain of cysteine (C)
Maleimide-activated—perform reactions at pH 6.5 to 7.5 in buffers such as PBS
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—must be dissolved in DMSO or DMF before further dilution in aqueous buffers
Medium length—spacer arm (total length added to target) is 32.6 angstroms; contains cyclohexane ring, which stabilizes adjacent maleimide

BMCC-Biotin is a maleimido-biotin compound for labeling protein cysteines and other molecules that contain sulfhydryl groups. This reagent specifically reacts with reduced thiols (-SH) in near-neutral buffers to form permanent (irreversible) thioether bonds. The unique feature of BMCC-Biotin is its spacer arm cyclohexane ring; this has a stabilizing effect that minimizes hydrolysis and degradation of the maleimide group until it has opportunity to conjugate with target thiols.

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

Biotinylation reagents differ in reactivity, length, solubility, cell permeability and cleavability. Three types of sulfhydryl-reactive compounds are available: maleimido, iodoacetyl and pyridyldithiol. Maleimide reagents specifically react with sulfhydryl groups (-SH) in near-neutral buffers to form permanent thioether bonds.

In proteins, sulfhydryls exist where there are cysteine (C) residues. Cystine disulfide bonds must be reduced to make sulfhydryl groups available for labeling. Hinge-region disulfide bridges of antibodies can be selectively reduced to make functional half-antibodies that can be labeled.

Alexa Fluor™ 350 Hydrazide (Invitrogen™)

Alexa Fluor® 350 Hydrazide is useful as a cell tracer and as a reactive dye for labeling aldehydes or ketones in polysaccharides or glycoproteins. Alexa Fluor® 350 is a blue fluorescent dye with excitation ideally suited to the UV laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 350 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 350 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

Detailed information about this AlexaFluor® hydrazide:

• Fluorophore label : Alexa Fluor® 350 dye
• Reactive group: hydrazide
• Reactivity: Aldehydes or keytones in polysaccharides or glycoproteins
• Ex/Em of the conjugate: 345/445 nm
• Extinction coefficient: 13,000 cm-1M-1
• Spectrally similar dyes: DAPI
• Molecular weight: 349.29

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. 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)

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.

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

Qdot™ 605 Biotin Conjugate Kit (Invitrogen™)

The biotin-labeled Qdot® 605 nanocrystals are available for detecting streptavidin probes or for creating noncovalent conjugates with streptavidin-labeled molecules or with other biotinylated molecules using a streptavidin bridge. The product is provided as 250 µL of a 2 µM solution and includes 30 mL of Qdot® incubation buffer.

Click-iT™ Biotin sDIBO Alkyne (Invitrogen™)

Click-iT Biotin sDIBO Alkyne reacts with azides via a copper-free Click chemistry reaction to produce biotin 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

Learn more about avidin-biotin detection ›

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.

LanthaScreen™ Amine Reactive Tb Chelate

As part of the LanthaScreen® TR-FRET toolbox of assay reagents, LanthaScreen® Amine Reactive Tb Chelate labeling reagent is available for assay development. The amine-reactive, isothiocyanate group conjugates to virtually any peptide or protein that contains one or more accessible amine moieties, such as the amine group found on a lysine residue or an unmodified N-terminal amine group of a peptide or protein. Amine-modified DNA oligonucleotides can be labeled as well. Energy transfer from the terbium donor to a suitable acceptor such as fluorescein is readily detected by monitoring an increase in acceptor fluorescence intensity. See the user guide for application-based protocols for labeling peptides containing free amino groups, amine-modified oligonucleotides, and IgG antibodies.

PowerLoad™ Concentrate, 100X (Invitrogen™)

PowerLoad™ is an optimized formulation of nonionic, Pluronic® surfactant polyols for the solubilization of water-insoluble dyes and other materials in physiological media. These surfactants, for instance Pluronic® F-127, have been used to help disperse acetoxymethyl (AM) esters of fluorescent ion indicators such as fluo-4, fura-2, indo-1, fluo-3, and SBFI; they appear to be required for loading of other dyes (e.g. SBFI-AM or PBFI-AM). The use of PowerLoad™ is optional with red shifted calcium indicators and other large molecular weight AM ester dyes, and may also be useful for dispersing other lipophilic probes. The concentration of Pluronic® surfactants in PowerLoad™ is less than 0.2%. PowerLoad™ is effective in combination with water soluble Probenecid (P36400) to aid AM ester dye-loading and retention in cells that actively extrude the de-acetylated form through anion pumps. Together, these reagents allow for maximal loading of dyes with a minimum of effort in both imaging and high throughput screening (HTS) applications. Appropriate controls should be performed to make certain that PowerLoad™ is not altering the membrane properties of the cell.

GlycanAssure™ APTS Kit (Applied Biosystems™)

The GlycanAssure™ APTS Kit is part of the GlycanAssure™ Glycan Analysis and Quantitation System and contains all reagents and buffers needed to analyze and quantitate N-glycans from glycoprotein samples. The GlycanAssure APTS N-glycan sample prep method consists of rapid deglycosylation using PNGase-F enzyme followed by magnetic bead-based glycan purification, glycan labeling with ATPS dye, and excess dye removal. APTS is a traditional dye used for glycan labeling for the past several years, and the labeled glycans can be analyzed using capillary electrophoresis (CE) or liquid chromatography (LC) system. The GlycanAssure sample prep workflow does not include time-consuming vacuum centrifugation steps or the use of toxic sodium cyanoborohydride like more traditional workflows, resulting in a streamlined, automatable method for the processing and analysis of 96 samples in ~9 hrs (< 4 hrs hands-on time).

The GlycanAssure APTS kit contains:
• GlycanAssure core reagents
• GlycanAssure beads
• GlycanAssure APTS labeling reagents
• GlycanAssure kits user guide

GlycanAssure Glycan Analysis and Quantitation System
The GlycanAssure Glycan Analysis and Quantitation System is the first glycan analysis system that provides both high throughput and high data quality through an integrated glycan analysis platform that helps save labor, time, and cost of analysis. The GlycanAssure system offers simple and easy magnetic bead-based sample preparation with multiple fluorescent dyes for glycan labeling, multi-capillary Sanger sequencing instruments for high-throughput CE-LIF-based glycan analysis, and assay-specific software for fast data analysis and reporting.

As part of the GlycanAssure Glycan Analysis and Quantitation System, the 3500 Genetic Analyzer for Protein Quality Analysis and 3500xL Genetic Analyzer for Protein Quality Analysis are multi-capillary CE instruments that enable parallel analysis of 8 or 24 samples on a 50-cm capillary array for high-throughput and high-resolution glycan analysis. The ability to analyze samples in parallel avoids the need for shortened run times to achieve high throughput. The 3500 Series systems for protein quality analysis come with integrated assay-specific GlycanAssure data acquisition and data analysis software for optimum performance and user experience.

Alexa Fluor™ 750 C5 Maleimide (Invitrogen™)

Alexa Fluor® 750 is a bright, near-infrared fluorescent dye with excitation ideally suited for the 633 nm laser line or dye-pumped laser excitation. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 750 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 750 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The maleimide derivative of Alexa Fluor® 750 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor® 750 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® maleimide:

Fluorophore label: Alexa Fluor® 750 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 753/783 nm
Extinction coefficient: 290,0000 cm-1M-1
Spectrally similar dyes: Cy7
Molecular weight: ~1350

Typical Conjugation Reaction
The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation.

The Alexa Fluor® maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

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

Thermo Scientific EZ-Link Hydrazide-LC-Biotin is a mid-length, simple, hydrazide-activated biotinylation reagent for labeling glycoproteins and other carbohydrate-containing compounds having oxidizable sugars or aldehydes.

Features of EZ-Link Biotin-LC-Hydrazide:

Glycoprotein labeling—biotinylate glycosylated proteins at sialic acid residues for detection or purification using streptavidin probes or resins
Cell surface labeling—biotinylate and isolate cell surface glycoproteins
Aldehyde-reactive—reacts with aldehydes formed by periodate-oxidation of sugar groups
Hydrazide-activated—perform reactions at pH 4 to 6 in buffers such as sodium acetate
Irreversible—forms semi-permanent hydrazone bonds; spacer arm cannot be cleaved
Solubility—usually dissolved in DMSO before further dilution in aqueous buffers
Spacer arm length—24.7Å

This biotin hydrazide reagent enables simple and efficient biotin labeling of polyclonal antibodies and other glycoproteins. Mild oxidation of antibodies with sodium periodate produces reactive aldehydes on the carbohydrate moieties of the Fc portion that can be modified by hydrazides. This approach is advantageous for labeling antibodies because biotinylation occurs only at the sites of glycosylation, which are primarily in the Fc region of the antibody, far from the antigen binding site.

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. Hydrazides and alkoxyamines are two types of carbonyl-reactive groups. Hydrazides (—NH-NH2) react specifically with aldehyde groups in slightly acidic conditions to form hydrazone linkages; these can be further reduced to stable secondary amine bonds using sodium cyanoborohydride (Part No. 44892). The reaction is more efficient in the presence of aniline (Part No. 88944). Alternatively, hydrazides can be conjugated to carboxylic acids using EDC carbodiimide chemistry.

Reactive aldehyde groups can be generated in glycoproteins and other polysaccharide compounds by oxidation of constituent sugar diols using sodium periodiate (Part No. 20504). Sialic acid residues are common components of protein glycosylation and are easily converted to aldehydes with 1 mM NaIO4.

Related Products
EZ-Link™ Hydrazide-Biotin

Click-IT™ GalNAz Metabolic Glycoprotein Labeling Reagent (Tetraacetylated N-Azidoacetylgalactosamine) (Invitrogen™)

The Click-iT® GalNAz metabolic glycoprotein labeling reagent provides the first part of a simple and robust two-step technique to identify and characterize cell surface O-linked glycoproteins. In step one, cultured cells are incubated with the azide-modified galactosamine (GalNAz). The azido-sugar is metabolically incorporated into cell surface O-linked glycoproteins through the permissive nature of the oligosaccharide biosynthesis pathway. In step two, via the chemoselective ligation or click reaction between an azide and an alkyne, the azido-labeled glycoproteins can then be detected with a Click-iT® Glycoprotein Detection Kit for gels (TAMRA or Dapoxyl® alkyne) or Western blots (biotin alkyne). These Click-iT® products are compatible with LC-MS⁄MS and Multiplexed Proteomics™ technologies for in-depth analyses of the glycoproteome.

SAIVI™ Alexa Fluor™ 647 Antibody/Protein 1 mg-Labeling Kit (Invitrogen™)

The SAIVI Alexa Fluor® 647 Antibody/Protein 1 mg-Labeling Kit provides a convenient means to label proteins with Alexa Fluor® 647 near-IR emitting dye. The kit is optimized for labeling and purifying 1 mg of IgG per conjugation reaction; comparable amounts of other proteins (>30 kDa) can also be labeled. To conveniently control the degree of labeling (DOL), this kit includes a DOL modulating reagent and instructions for decreasing the DOL from its intrinsic highest value. Using this method, protein preparations with varying ratios of dye to protein can be quickly and reproducibly obtained, allowing more efficient optimization in applications such as in vivo imaging, where the DOL of a protein can have significant effects on the signal-to-background ratio, biodistribution, and clearance.

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™ 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.

DyLight™ 633 Microscale Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 633 Microscale Antibody Labeling Kit contains an NHS ester-activated derivative of high-performance DyLight 633 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG—the amine-reactive DyLight 633 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

DyLight 633 fluoresces red and has physical properties comparable to other 633 dyes, including Alexa Fluor™ 633. 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 633 Amine-Reactive Dye is available as a stand-alone reagent.

Features of DyLight 633 NHS Ester:

High performance—DyLight 633 shows brighter fluorescence than Alexa Fluor 633
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Convenient kit sizes—standard and microscale sizes are offered to match your experimental needs
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 633 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 633 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 633 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

Related Products
DyLight™ 633 NHS Ester
DyLight™ 633 Antibody Labeling Kit

Alexa Fluor™ 555 Microscale Protein Labeling Kit (Invitrogen™)

Microscale Protein Labeling Kits provide a convenient means for attaching a fluorescent label to a small amount of antibody or protein (20–100 µg). The kits are available in four Alexa Fluor® colors (or biotin) and supply everything needed for three labeling and separation reactions.

Important Features of Microscale Protein Labeling Kits:
• Labeled proteins typically ready to use typically in 2 hours (~30 minutes hands-on time)
• Optimized for 20–100 µg of protein with molecular weights between 12 and 150 kDa
• Purified using convenient spin filters with yields between 60 and 90%
• Stabilizing proteins must be removed from the sample before labeling

Stable Reaction Chemistry and Superior Alexa Fluor® Dyes
In the Microscale Protein Labeling Kits, the reactive dye contains a succinimidyl (NHS) ester moiety that reacts with primary amines of proteins to form stable dye-protein conjugates. 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.

Pierce™ Antibody Biotinylation Kit for IP (Thermo Scientific™)

The Thermo Scientific™ Pierce™ Antibody Biotinylation Kit for IP provides biotinylation reagents designed specifically for the labeling of primary antibodies used in immunoprecipitation applications.

Optimized for IP—reagents and protocols developed specifically to address antibody labeling for immunoprecipitation applications
Easy to use—kit configuration specifically optimized to accommodate 50–200 μg of antibody in a 100 μL reaction; desalting columns are provided to remove excess biotinylation reagent after labeling
Amine reactive—reacts with primary amines (-NH2) of antibodies
Enhanced solubility—pegylation improves water solubility of the biotinylated antibody and prevents aggregation upon storage
Reduced steric hindrance—longer spacer arm (29 angstroms) minimize steric hindrance when binding to avidin molecules

The Pierce Antibody Biotinylation Kit reagents have been optimized and validated to biotin label antibodies for IP and co-IP reactions. Determining the optimal number of biotins to attach to the target molecule is one of the major challenges of biotinylation. For IP and co-IP applications, too many biotins result in reduced affinity for the target antigen, while too few biotins result in antibody leaching upon elution of the target antigen. The biotin labeling procedure in the Pierce Antibody Biotinylation Kit for IP has been developed to address this challenge.

The kit contains sufficient reagents to label 50–200 µg of antibody in 100 µL reaction volumes for eight samples. The NHS-PEG4-biotin labeling reagent contains an amine-reactive N-hydroxysuccinimide ester (NHS) group and a water-soluble PEG4 spacer for optimal labeling and is provided in easy-to-use, single-use microtubes. Both the labeling efficiency of the biotinylation reagent and binding affinity of the labeled antibody have been validated using mouse monoclonal (IgG1, IgG2), rabbit polyconal, and rabbit monoclonal antibodies. Zeba™ Desalting Spin Columns are provided for easy and efficient removal of salts and excess biotin.

Applications:
• IP and co-IP
• IP and co-IP for mass spectrometry applications
• Antibody attachment to streptavidin agarose and magnetic supports

DyLight™ 594 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 594 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 594 used to fluorescently label sulfhydryl-containing peptides, proteins and other biomolecular probes.

DyLight 594 provides vibrant orange-to-red fluorescence with better performance than other rhodamine derivatives including Alexa Fluor™ 594 and Texas Red™ dye for fluorescent applications over a broad pH range (pH 4-9). The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates.

Features of DyLight 594 Maleimide:

High performance—DyLight 594 shows brighter fluorescence than Alexa Fluor 594 and Texas Red
Specific—maleimide-activated dye labels proteins and other molecules at reduced sulfhydryls (-SH)
Efficient labeling methods—well-characterized chemistry and optimized protocols provide for reliable, high-quality labeling
Optimized antibody labeling procedure—complete protocol for IgG reduction and labeling and calculating the labeling efficiency

Applications:
• Antibody labeling for immunofluorescence applications, including immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting and ELISA assay
• Target macromolecule labeling for in vitro and in vivo fluorescent detection strategies

DyLight 594 Sulfhydryl-Reactive Dye is activated with a maleic acid imide (maleimide) moiety to form a reactive alkylation reagent. Labeling occurs through reaction of the maleimide-activated dye with reduced sulfhydryl groups (-SH) to form stable thioether bonds. Maleimides are specific for sulfhydryl groups between pH 6.5-7.5. Learn more about maleimide chemistry.

APEX™ Biotin-XX Antibody Labeling Kit (Invitrogen™)

The APEX® Antibody Labeling Kits are our best option for covalently attaching a fluorophore to small amounts of IgG antibody (~10–20 μg). It is ideal for the efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Labeled antibodies are ready for use in imaging or flow cytometry applications in as little as 2.5 hours with very little hands on time.

Important Features of Alexa Fluor® APEX® Antibody Labeling Kits:

• Labeled antibodies typically ready to use in 2.5 hours (~15 minutes hands on time)
• Designed to label 10–20 μg of IgG
• Covalent attachment
• Compatible with contaminating proteins or stabilizers like BSA
• No columns needed; everything you need is supplied for 5 separate labelings
• Choose from Alexa Fluor® 488, 555, 568, 594, and 647 dyes, Oregon Green® 488 dye, Pacific Blue™ dye, and Biotin-XX.


Better Results and Workflows With Primary Labeled Antibodies
A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding. Further, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment if they are directly labeled with compatible fluorophores.

Contaminating Proteins or Protein Stabilizers Are Not a Problem
Many IgG antibodies are often available only in small quantities and packaged with stabilizing proteins, such as BSA, or other contaminants which can interfere with the amine-reactive labeling reagents. The APEX® Antibody Labeling Kits avoids this by utilizing a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX® antibody labeling tip. Contaminants are simply eluted through the tip, prior to applying the amine-reactive label.

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.

Alexa Fluor™ 660 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.

Zenon™ Alexa Fluor™ 647 Mouse IgG2a Labeling Kit (Invitrogen™)

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

Important Features of Zenon® Labeling Technology:

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


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

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

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

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

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

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

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

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

Related Links:

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

DyLight™ 515-LS NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight Long Stokes Shift Specialty Dyes have fluorescence emission from green to far red with a single excitation peak that matches the 488 laser line and very large Stokes shifts that inhibit quenching. DyLight 515-LS dye has excitation and emission peaks at 519 and 648 nm, respectively.

DyLight Long Stokes Shift Specialty Dyes are a family of labeling agents that provide bright fluorescent detection for multiplexed imaging. The dyes share a common excitation range, while their emission properties extend from the green to the far red wavelengths. The dyes are based on a core coumarin structure that is modified to emit at different wavelengths. Each dye contains an amine-reactive NHS ester for rapid modification of antibodies, proteins, peptides or other biomolecules through amide bond formation. DyLight Long Stokes Shift Dyes can be used individually or combined with DyLight 488 for multiplexed analysis. The excitation and emission peaks are sufficiently separated so as to minimize dye-dye quenching in assays.

General features of the DyLight Long Stokes Shift Specialty Dyes:

Single excitation wavelength—DyLight Long Stokes Shift Dyes all can be excited together at 488 nm using an Argon laser
Image multiple targets simultaneously—the four Long Stokes Shift Dyes can be used with DyLight 488 for multiplex fluorescence applications
NHS ester reactive group—allows immediate labeling of antibodies, proteins, peptides and other amine-containing molecules through amide bond formation
Multiple solubility options—choose from hydrophilic to hydrophobic dyes to optimize the right dye label for the best performance in an application
Large Stokes shift inhibits quenching—minimal overlap between excitation and emission peaks inhibits dye-dye quenching effects
Use in FRET-based assays—the extremely broad separation between excitation and emission peaks allow for use of the Long Stokes Shift Dyes in energy transfer applications where the acceptor dye is spectrally far removed from the excitation wavelength.

Criteria to consider when choosing a DyLight Long Stokes Shift Specialty Dye:

• Excitation and emission wavelengths—all dyes share a common excitation wavelength range with options for emission from the green to the far red wavelengths.
• Water solubility—DyLight Long Stokes Shift Dyes can be initially solubilized in methanol, ethanol, DMF or DMSO with transfer into an aqueous buffer medium for labeling biomolecules

Applications:
• Quenchers and FRET pairs
• Conjugation to silica particles
• STED microscopy
• Multicolor microscopy
• 4Pi microscopy
• Dual-color 3D nanoscopy
• Dynamic light scattering
• Graded stokes shift
• Duplex enhanced RT-PCR
• High-resolution optical microscopy

Related Products
DyLight™ 485-LS NHS Ester
DyLight™ 510-LS NHS Ester
DyLight™ 521-LS NHS Ester