Antibody-Labeling

Each immunolabeling experiment presents its own challenges. From the initial binding of the primary antibody to the final detection of the fluorophore label—and at every wash step in between—you face optimization protocols that will determine the sensitivity and selectivity of your experiment. Direct labeling of your primary antibody does provide two key advantages:

  • Multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment
  • Primary antibodies directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding than labeled secondary antibodies.

We offer a range of antibody labeling kits to choose from. The one you select ultimately depends on the requirements of your application. Use the selection tables below to help you decide on your best option.

Which antibody labeling kit do you need?

These kits have the easiest protocols and require the least amount of hands-on experience.

  Zip Rapid Antibody Labeling Kits Zenon Antibody Labeling Kits
Amount IgG labeled/reaction* 100 µg 1–20 µg
Label target/method Free lysines/covalent amine-reactive chemistry Fc portion of IgG/antibody affinity
Compatible with BSA or other stablizers in sample buffer? No Yes
Can measure degree of labeling? No No
Can store conjugate longer than 24 hr? Yes No
Requires post-label purification? No No
Time required for protocol 15 min 10 min
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These kits have quick protocols and/or require minimal hands-on time.

  APEX Antibody Labeling Kits Zip Rapid Antibody Labeling Kits Zenon Antibody Labeling Kits
Amount IgG labeled/reaction* 10–20 µg 100 µg 1–20 µg
Label target/method Free lysines/covalent amine-reactive chemistry Fc portion of IgG/antibody affinity
Can measure degree of labeling? No No No
Can store conjugate more than 24 hr? Yes Yes No
Site specific label? No No Yes
Compatible with BSA or other stablizers in sample buffer? Yes No Yes
Requires post-label purification? No No No
Time required for protocol 2 hr 15 min 10 min
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These kits use the standard covalent, amine-reactive dyes that randomly label lysines on the antibody.

  APEX Antibody Labeling Kits Antibody Labeling Kits Zip Rapid Antibody Labeling Kits Protein Labeling Kits
Amount IgG labeled/reaction* 10–20 µg 100 µg 100 µg 1 mg
Label target/method Free lysines/covalent amine-reactive chemistry
Can measure degree of labeling? No Yes No Yes
Can store conjugate more than 24 hr? Yes Yes Yes Yes
Site specific label? No No No No
Compatible with BSA or other stablizers in sample buffer? Yes No No No
Requires post label purification? No Yes No Yes
Time required for protocol 2 hr 90 min 15 min 2 hr
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These kits allow labeling as little as 1 µg in one reaction with minimal loss of conjugate.

  APEX Antibody Labeling Kits Zenon Antibody Labeling Kits
Amount IgG labeled/reaction* 10–20 µg 1–20 µg
Label target/method Free lysines/covalent amine-reactive chemistry Fc portion of IgG/antibody affinity
Can measure degree of labeling? No No
Can store conjugate more than 24 hr? Yes No
Site specific label? No Yes
Compatible with BSA or other stablizers in sample buffer? Yes Yes
Requires post label purification? No No
Time required for protocol 2 hr 10 min
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These methods prevent labeling of the antigen binding site.

  Zenon Antibody Labeling Kits SiteClick Antibody Labeling Kits
Amount IgG labeled/reaction* 1–20 µg 100 µg to 5 mg
Label target/method Fc portion of IgG/antibody affinity Sugars on IgG heavy chain/covalent, copper-free click chemistry
Can measure degree of labeling? No Yes
Can store conjugate more than 24 hr? No Yes
Site specific label? Yes Yes
Compatible with BSA or other stablizers in sample buffer? Yes Yes
Requires post-label purification? No Yes
Time required for protocol 10 min overnight incubation
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

For applications where it is critical to get the exact same conjugate every time.

  SiteClick Antibody Labeling Kits
Amount IgG labeled/reaction* 100 µg to 5 mg
Label target/method Sugars on IgG heavy chain/covalent, copper-free click chemistry
Can measure degree of labeling? Yes
Can store conjugate more than 24 hr? Yes
Site-specific label? Yes
Compatible with BSA or other stablizers in sample buffer? Yes
Requires post-label purification? Yes
Time required for protocol overnight incubation
Optimal applications** FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye: protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These kits have the easiest protocols and require the least amount of hands-on experience.

  Zip Rapid Antibody Labeling Kits Zenon Antibody Labeling Kits
Amount IgG labeled/reaction* 100 µg 1–20 µg
Label target/method Free lysines/covalent amine-reactive chemistry Fc portion of IgG/antibody affinity
Compatible with BSA or other stablizers in sample buffer? No Yes
Can measure degree of labeling? No No
Can store conjugate longer than 24 hr? Yes No
Requires post-label purification? No No
Time required for protocol 15 min 10 min
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These kits have quick protocols and/or require minimal hands-on time.

  APEX Antibody Labeling Kits Zip Rapid Antibody Labeling Kits Zenon Antibody Labeling Kits
Amount IgG labeled/reaction* 10–20 µg 100 µg 1–20 µg
Label target/method Free lysines/covalent amine-reactive chemistry Fc portion of IgG/antibody affinity
Can measure degree of labeling? No No No
Can store conjugate more than 24 hr? Yes Yes No
Site specific label? No No Yes
Compatible with BSA or other stablizers in sample buffer? Yes No Yes
Requires post-label purification? No No No
Time required for protocol 2 hr 15 min 10 min
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These kits use the standard covalent, amine-reactive dyes that randomly label lysines on the antibody.

  APEX Antibody Labeling Kits Antibody Labeling Kits Zip Rapid Antibody Labeling Kits Protein Labeling Kits
Amount IgG labeled/reaction* 10–20 µg 100 µg 100 µg 1 mg
Label target/method Free lysines/covalent amine-reactive chemistry
Can measure degree of labeling? No Yes No Yes
Can store conjugate more than 24 hr? Yes Yes Yes Yes
Site specific label? No No No No
Compatible with BSA or other stablizers in sample buffer? Yes No No No
Requires post label purification? No Yes No Yes
Time required for protocol 2 hr 90 min 15 min 2 hr
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These kits allow labeling as little as 1 µg in one reaction with minimal loss of conjugate.

  APEX Antibody Labeling Kits Zenon Antibody Labeling Kits
Amount IgG labeled/reaction* 10–20 µg 1–20 µg
Label target/method Free lysines/covalent amine-reactive chemistry Fc portion of IgG/antibody affinity
Can measure degree of labeling? No No
Can store conjugate more than 24 hr? Yes No
Site specific label? No Yes
Compatible with BSA or other stablizers in sample buffer? Yes Yes
Requires post label purification? No No
Time required for protocol 2 hr 10 min
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

These methods prevent labeling of the antigen binding site.

  Zenon Antibody Labeling Kits SiteClick Antibody Labeling Kits
Amount IgG labeled/reaction* 1–20 µg 100 µg to 5 mg
Label target/method Fc portion of IgG/antibody affinity Sugars on IgG heavy chain/covalent, copper-free click chemistry
Can measure degree of labeling? No Yes
Can store conjugate more than 24 hr? No Yes
Site specific label? Yes Yes
Compatible with BSA or other stablizers in sample buffer? Yes Yes
Requires post-label purification? No Yes
Time required for protocol 10 min overnight incubation
Optimal applications** FC, IF, WB, HCA FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye:protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

For applications where it is critical to get the exact same conjugate every time.

  SiteClick Antibody Labeling Kits
Amount IgG labeled/reaction* 100 µg to 5 mg
Label target/method Sugars on IgG heavy chain/covalent, copper-free click chemistry
Can measure degree of labeling? Yes
Can store conjugate more than 24 hr? Yes
Site-specific label? Yes
Compatible with BSA or other stablizers in sample buffer? Yes
Requires post-label purification? Yes
Time required for protocol overnight incubation
Optimal applications** FC, IF, WB, HCA
*Kits are optimized for whole IgG protein, molecular weight 150 kDa unless otherwise indicated. For those kits, please see instructions on how to adjust protein concentration for optimal dye: protein ratios.
**FC=flow cytometry; IF=immunofluorescence; WB=western blot analysis; HCA=high content analysis

Not looking for a kit or need a different dye?

Find standalone amine-reactive dyes and more information about amine-reactive chemistries in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1, The Molecular Probes Handbook.

We also have thiol-reactive dyes available as standalone reagents and you can learn more about these in the Thiol-Reactive Probes—Chapter 2, The Molecular Probes Handbook.

Covalent antibody labeling

Zip Rapid Antibody Labeling Kits

Zip Rapid Antibody Labeling Kits

Zip Alexa Fluor Rapid Antibody Labeling Kits allow you to efficiently label your precious antibody with fluorescent dyes to create an antibody conjugate that is ready to use within 15 minutes.

  • Label 100 μg of IgG antibody
  • Three labeling reactions per kit
  • Very fast and easy to use
  • 100% antibody recovery—no purification steps
  • No chemistry or conjugation experience needed

The kit contains everything you need to perform three separate labeling reactions. Covalently labeled conjugates are ideal for multiple applications, including flow cytometry, fluorescence microscopy, immunohistochemistry, primary antibody detection, ELISAs, immunocytochemistry, and indirect FISH. Simply add 1 mL water to component A containing Zip buffer, add 1 mg of antibody, incubate for 15 minutes and your antibody is ready to use (Figure 1).

Zip Rapid Antibody Labeling Kit overview
 Click image to enlarge

Figure 1. Zip Rapid Antibody Labeling Kit overview.

Zip Rapid Antibody Labeling Kits selection guide

Label Ex/Em* Cat. No.
Alexa Fluor 488 494/519 Z11233
Alexa Fluor 555 555/565 Z11234
Alexa Fluor 647 650/668 Z11235

Ex/Em = Fluorescence excitation and emission maxima, in nm.

Find more information about Alexa Fluor dyes and other fluorophores in the Fluorophore Selection Guide

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Apex Antibody Labeling Kits

Apex Antibody Labeling Kits

Our Apex Antibody Labeling Kits utilize a unique solid-phase labeling method that enables an efficient, convenient, and reliable method for covalently attaching fluorescent labels to very small amounts of IgG antibody.

  • Labels 10–20 µg of IgG antibody per labeling reaction
  • Five labeling reactions per kit
  • Can be used with buffers containing BSA or other stabilizers and contaminants
  • Typical labeled antibody yield is 40-80%

The APEX Antibody Labeling Kits use a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX antibody labeling tip (Figure 2). Any contaminants, including stabilizing proteins or amine-containing buffers, are simply eluted through the tip. After applying the amine-reactive label, a fluorescent IgG conjugate is ready for use in any imaging or flow cytometry application in as little as 2.5 hours with very little hands-on time. The typical yield of labeled antibody using this method ranges from 40–80%.

Each APEX Antibody Labeling Kit includes all reagents required to perform 5 separate labeling reactions of 10–20 μg of IgG antibody. See the table below for the available fluorophores or haptens.  

275-wide.par.26956.image.243.301.1.mp10468-apex-antibody-labeling-gif.mp10468-apex-antibody-labeling-gif

Figure 2. APEX antibody labeling tip.

APEX Antibody Labeling Kits selection guide

Label Ex/Em (nm) Cat. No.
Alexa Fluor 488 496/519 A10468
Alexa Fluor 555 555/565 A10470
Alexa Fluor 568 578/603 A10494
Alexa Fluor 594 590/617 A10474
Alexa Fluor 647 650/665 A10475
Oregon Green 488 496/524 A10476
Pacific Blue dye 416/451 A10478
Biotin-XX* NA A10495
* The biotin-XX provides a 14-atom spacer between the antibody and the biotin moiety.

Ex/Em = Fluorescence excitation and emission maxima, in nm.

Find more information about Alexa Fluor dyes and other fluorophores in the Fluorophore Selection Guide

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Antibody Labeling Kits

Antibody Labeling Kits

Thermo Fisher Scientific Antibody Labeling Kits label 100 µg IgG polyclonal and monoclonal antibodies with our superior Alexa Fluor, Pacific Blue, or Pacific Orange dyes.

  • Label 100 µg
  • Five labeling reactions per kit
  • Labeled antibodies ready in 90 minutes
  • ~15 minutes actual hands-on time
  • Requires removal of BSA and other protein stabilizers prior to labeling reaction

The Antibody Labeling Kits utilize an amine-reactive Alexa Fluor, Pacific Blue or Pacific Orange fluorophore to covalently attach the label to the IgG antibody of interest. Once formed, the covalent bond between the label and the protein is extremely stable and you are using the same chemistry we use to prepare our own primary and secondary conjugates.

These kits are optimized for labeling 100 µg of antibody per reaction. Simply adjust the protein concentration to ~1 mg/mL in the provided buffer, then add it to a vial of amine-reactive dye. Any stabilizing proteins (e.g., BSA) must be removed prior to the labeling reaction.

Purification is accomplished on a size exclusion spin column optimized for ≥40 kDa proteins (Figure 3). The entire labeling and purification procedure can be completed in as little as 90 minutes; everything needed to perform five conjugations is provided.

How Antibody Labeling Kits work

Figure 3. Overview of Antibody Labeling Kits process. Simply adjust the protein concentration to ~1 mg/mL in the provided buffer, then add it to a vial of amine-reactive dye. Purification is accomplished on a size exclusion spin column optimized for ≥40 kDa proteins.

Antibody Labeling Kits selection guide

Ex/Em = Fluorescence excitation and emission maxima, in nm.

Find more information about Alexa Fluor dyes and other fluorophores in the Fluorophore Selection Guide

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Protein Labeling Kits

Protein Labeling Kits

Thermo Fisher Scientific Protein Labeling Kits provide a nearly effortless way to covalently label 1 mg of IgG antibody with a fluorescent dye (including Alexa Fluor dyes) or biotin.

  • Label 1 mg antibody
  • Three labeling reactions in each kit
  • Removal of BSA and other stablizers is required prior to labeling
  • Not compatible with amine-containing buffers (e.g. Tris)
  • Ready to use in 2 hours
  • ~ 30 minutes actual hands-on time

The Protein Labeling Kits utilize an amine-reactive fluorophore or hapten to covalently attach the label to the IgG antibody. Once formed, the covalent bond between the label and the protein is extremely stable—you are using the same chemistry we use to prepare our own primary and secondary conjugates.

Simply add ~1 mg of purified IgG (in ~500 μL and free of amine-containing buffers such as Tris) to one of the vials containing a premeasured quantity of amine-reactive dye and a magnetic stir bar. Purification is accomplished on a gravity-feed size exclusion column supplied with the kit (Figure 4). The entire labeling and purification procedure can be completed in as little as 2 hours; everything needed to perform three conjugations is provided.

Protein Labeling Kits workflow

Figure 4. Protein Labeling Kits are the simplest way to label 1 mg of IgG antibody.

Protein Labeling Kits selection guide

Ex/Em = Fluorescence excitation and emission maxima, in nm.

Find more information about Alexa Fluor dyes and other fluorophores in the Fluorophore Selection Guide

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Site-specific antibody labeling

Affinity labeling with Zenon Antibody Labeling Kits

Zenon Antibody Labeling Kits

Invitrogen Zenon labeling technology provides a rapid and reliable method for producing fluorescently labeled antibodies using a variety of fluorochromes, even with small amounts of unpurified starting material such as hybridoma culture supernatant. Zenon fragments are specifically designed to target and bind to the Fc portion of the primary antibody only, giving a rapid, noncovalent method of quickly labeling small quantities of primary antibody. Antibodies labeled using Zenon labeling technology are suitable for use in all applications where a directly labeled antibody can be used. Advantages of Zenon labeling technology include:

  • 10 minute labeling time
  • 1–20 µg primary antibody per labeling
  • Compatible with BSA and other stabilizing proteins
  • No purification step
  • Multiplex with other mouse monoclonal antibodies simultaneously

Zenon labeling technology provides a versatile, easy-to-use system for labeling human, mouse IgG1, IgG2a, and IgG2b antibodies and rabbit IgG antibodies. Invitrogen Zenon fragments are specifically designed to target and bind to the Fc portion of the primary antibody only, giving a rapid, noncovalent method of quickly labeling small quantities of primary antibody (Figure 6). Zenon labeling technology is simple and efficient; the entire labeling procedure takes only 10 minutes.

Once the Zenon–antibody complex has formed, just prior to use, nonspecific IgG is added to block any unbound Zenon fragments, making column purification unnecessary. The Zenon complex can then be applied directly to the sample.

Antibodies labeled using Zenon labeling technology display fluorescence intensity or enzymatic activity similar to that observed for directly labeled antibody conjugates (Figure 7).

How Zenon Labeling Technology works
 Click image to enlarge

Figure 6. How Zenon labeling technology works. Noncovalent labeling with Zenon antibody labeling (top) vs. covalent labeling with the APEX, Microscale, Antibody, Protein, Zip Rapid and SAIVI Labeling Kits (bottom).

Zenon labeling technology
 Click image to enlarge

Figure 7. Zenon labeling technology produces antibody conjugates with brightness comparable to or better than those obtained from direct conjugates. Antibodies to various lymphocyte markers were labeled either covalently with allophycocyanin (APC) or noncovalently with 1 μg of a Zenon APC complex. Labeled antibodies were then used to stain samples of human peripheral blood lymphocytes. The brightness of the Zenon staining complex can be further enhanced by increasing the ratio of Zenon labeling technology reagent to the primary antibody used in the preparation of the complex.

Zenon Antibody Labeling Kits guide

Reactivity Target isotype Find products
Mouse IgG1 See all kits
IgG2a See all kits
IgG2b See all kits
Human IgG See all kits
Rabbit IgG See all kits

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Click chemistry labeling with SiteClick Antibody Labeling Kits

SiteClick Antibody Labeling System

Thermo Fisher Scientific SiteClick Labeling Kits allow simple and gentle site-selective attachment of detection molecules to heavy chain N-linked glycans—far from the antigen-binding domain—providing excellent reproducibility from labeling to labeling and from antibody to antibody. A number of different detection molecules can be site-selectively attached to the heavy chain glycans—including phycobiliproteins (e.g., R-PE), QDot probes, Alexa Fluor dyes , pHrodo dye, metal-chelating compounds, and other small molecules like biotin—allowing multiplex analysis with antibodies from the same species. Features include:

  • Labeling of 100 μg up to 5 mg of antibody
  • Easy to follow step-by-step protocol
  • Highly efficient, site-specific, reproducible labeling chemistry
  • Kits and dyes available in a flexible format for easy design of multi-color experiments

In general, IgG antibodies contain two N-linked glycans attached to specific conserved asparagine residues located in the antibody heavy chain Fc domain. These sugar chains, predominantly complex biantennary glycans with two terminal branches, are structurally quite homogeneous, and the terminal sequences of the glycan branches are highly consistent. Most of the antibody glycan branches terminate with galactose-N-acetylglucosamine (Gal-GlcNAc-) or with N-acetylglucosamine (GlcNAc-). Removal of the terminal Gal residue with β-galactosidase unmasks the majority of terminal GlcNAc labeling sites for the subsequent enzymatic β-galactosyl transferase (GalT) reaction (Figure 8). After cleavage of terminal Gal residues by β-galactosidase, each N-linked glycan will contain, on average, 2 terminal GlcNAc residues per heavy chain (4 terminal GlcNAc per antibody). The azide-activated antibody can now be labeled with a dibenzocyclooctyne (DIBO)-functionalized probe using copper-free click chemistry (Figure 9).

How to site-specifically label your antibody using SiteClick Technology

This video explains how to easily and site specifically label an antibody using an enzymatic and Click chemistry approach. This method can be applied to any intact IgG antibody and requires no antibody engineering or complex methodology.

Step 1 and 3 of the SiteClick antibody labeling process
 Click image to enlarge

Figure 8. The SiteClick Antibody Labeling Kits overview. (A) The first step in the SiteClick antibody labeling process involves removal of terminal galactose residues from the heavy chain N-linked glycans of the antibody using β-galactosidase, exposing essentially all possible modifiable GlcNAc residues. Second, the free terminal GlcNAc residues are activated with azide tags by enzymatic attachment of GalNAz to the terminal GlcNAc residues using the GalT(Y289L) enzyme. (B) In the third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-functionalized probe of choice (e.g., Alexa Fluor 488 sDIBO alkyne) (see Figure 9 for this reaction). The average degree of labeling is 3–3.5 labels per antibody.

Copper-less click chemistry azide/DIBO reaction

Figure 9. Copper-less click chemistry azide/DIBO reaction. The azide and alkyne moieties are interchangeable. The molecule can be labeled with a DIBO and reacted with a fluorophore or hapten-azide.

The SiteClick method is compatible with antibodies from a number of different species including, but not limited to, human, rabbit, mouse, rat, goat, hamster, and chicken. Additionally, SiteClick labeling is effective with several antibody classes such as IgG, IgM, and IgY; note that chicken IgY antibodies have 6 heavy chain glycans instead of 2 and therefore can be labeled to a higher extent.

SiteClick antibody labeling system offers flexibility

The SiteClick Antibody Labeling system is available in two formats:

  • Kits that contain both the azido modification components for preparing the antibody and a sDIBO alkyne to label the antibody (complete reaction featured in Figure 8A and B).
  • A kit that contains only the azido modification component (the first 2 steps of the reaction depicted in Figure 8A).
  • A variety of standalone sDIBO alkyne reagents that can be used to label the modified antibody (step in Figure 8B).

This flexibility of formats allows several advantages including the ability to easily change the fluorescent dye used, without impacting the degree of labeling of the same antibody and the ability to prepare a large batch of modified antibody and label only a small amount as needed, again, preserving the consistency of degree of labeling and functionality.

Antibody functionality is highly preserved

The covalent and site-specific attachment of the dyes to the N-linked glycans on the antibody heavy chain Fc domain ensures the immunogenicity of the antigen binding site is preserved, resulting in more efficient binding to the antigen as compared to antibodies randomly labeled with amine-reactive dyes. In a side-by-side comparison of SiteClick-labeled and amine-reactive labeled anti-troponin 1 antibody, the sensitivity of the SiteClick-labeled antibody was greater than that of the amine-reactive labeled antibody (Figure 10).

Comparison of SiteClick antibody labeling with amine-reactive antibody labeling in a bead-based sandwich assay

Figure 10. Comparison of SiteClick antibody labeling with amine-reactive antibody labeling in a bead-based sandwich assay. Anti-Troponin (Trpl) antibody capture beads were used and troponin antigen detection was performed using biotinylated anti-Trpl antibody labeled with either SiteClick system or amine-reactive system. Binding was detected using a streptavidin-mediated chemiluminescence assay.

Lot-to-lot consistency in antibody degree of labeling

Labeling antibodies with the SiteClick antibody labeling system gives you confidence that the antibody will be labeled the same way every time, with no time needed for reaction optimization. This means the same number of dyes attached to each molecule, the same preservation of the antigen binding site, providing in the same results every time. An example of the consistency in labeling between different antibodies is shown in Figure 11.

Lot-to- lot and antibody-to-antibody consistency

Figure 11. Lot-to- lot and antibody-to-antibody consistency. Twenty-eight different primary antibodies from different species were labeled by different users over a period of three years under the exact same conditions using the β-galactosidase-cleaved azide-activated antibodies and SiteClick Alexa Fluor 488 sDIBO Alkyne dye. The degree of labeling averaged 3.4+/- 0.39, demonstrating the consistency of the SiteClick labeling system. See Table 1 for the types, species and antigenicity of these antibodies.

Table 1. Antigenicity of primary antibodies labeled in degree of labeling study (Figure 11).

Human IgG1 Human IgG3 Mouse IgG1 Mouse IgG2a Goat Polyclonal
EGFR Lymphoma cell GD2 CD4 Apolipoprotein-A2
HER2/neu GD3 β-tubulin CD3  
sLea sLea CD8a CD8  
GD2 f-GM1 CD45 CD56  
GM2 Tn C3b, iC3b C3b, iC3b, C3dg  
J591   Interferon-γ    

SiteClick Antibody Labeling Kits selection guide

Product name Antibody quantity labeled Ex/Em * Cat. No.
Kits for antibody modification only
SiteClick Azido Modification Kit 100 μg N/A S20026
1 mg S10900
5 mg S10901
Standalone labeling reagents
SiteClick Alexa Fluor 488 sDIBO Alkyne 100 μg 495/519 C20027
1 mg S10904
5 mg S10909
Site Click Alexa Fluor 555 sDIBO Alkyne 100 μg 555/565 C20027
1 mg S10905
5 mg S10910
SiteClick Alexa Fluor 647 sDIBO Alkyne 100 μg 650/665 C20029
1 mg S10906
5 mg S10911
SiteClick iFL pHrodo Red sDIBO Alkyne 100 μg 560/585 C20034
1 mg S10903
5 mg S10908
SiteClick Biotin sDIBO Alkyne 100 μg N/A C20030
1 mg S10902
5 mg S10907
SiteClick Amine sDIBO Alkyne 100 μg N/A C20031
SiteClick SDP Ester sDIBO Alkyne 100 μg N/A C20032
Kits containing antibody modification + labeling reagent
SiteClick R-PE Antibody Labeling kit 100 μg 565/578 S10467
SiteClick Qdot 525 Antibody Labeling kit 100 μg 405/525 S10449
SiteClick Qdot 565 Antibody Labeling kit 100 μg 405/565 S10450
SiteClick Qdot 585 Antibody Labeling kit 100 μg 405/585 S10451
SiteClick Qdot 605 Antibody Labeling kit 100 μg 405/605 S10469
SiteClick Qdot 625 Antibody Labeling kit 100 μg 405/625 S10452
SiteClick Qdot 655 Antibody Labeling kit 100 μg 405/655 S10453
SiteClick Qdot 705 Antibody Labeling kit 100 μg 405/705 S10454
SiteClick Qdot 800 Antibody Labeling kit 100 μg 405/800 S10455

*Ex/Em = Fluorescence excitation and emission maxima, in nm.
Find more information about Alexa Fluor dyes and other fluorophores in the Fluorophore Selection Guide

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