Antibody Labeling Kits

The kit contains everything you need to perform three separate labeling reactions. Covalently labeled conjugates are exceptional 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).

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ReadyLabel Antibody Labeling Kits allow for fast, convenient antibody conjugation of off-the-shelf antibodies and cell culture supernatants without prior purification of the antibody solution. This works because the provided ReadyLabel spin columns contain resin that filter out contaminating stabilizer proteins present in the antibody solution (Figure 3). The resulting covalently bonded antibody conjugate can be used with flow cytometry, western blotting, ELISA, and indirect FISH.

ReadyLabel kits are equipped with your choice of our most popular stains to produce high yields of bright, soluble conjugates. Need a different dye? Consider our ReadyLabel Flex Kits, which contain everything needed except the dye, and use a dye of your choice. All our ReadyLabel kits are available in two different pack sizes, designed for labelings of 20 μg and 100 μg of IgG per reaction, and include all components necessary for five labeling reactions.

Figure 3. Antibodies are often stored with BSA or other protein stabilizers to prevent degradation. ReadyLabel kits are equipped with resin-containing spin columns that capture these contaminants and allow you to skip the purification step of your antibody solution. With approximately 20 minutes of hands-on time, and 30 minutes of incubation at room temperature, your covalently bonded antibody conjugate is now ready to use.

ReadyLabel Antibody Labeling Kits offer a unique advantage for multiplexing experiments. Unlike secondary detection methods, which are often constrained by the number of available IgG species, our kits allow you to directly conjugate your primary antibodies. This means you can achieve higher levels of multiplexing with greater flexibility.

Designed with high-quality, bright dyes and a focus on minimizing background noise, our kits enable clear and precise signals in your experiments. This clarity enhances the reliability and accuracy of your results, making ReadyLabel Antibody Labeling Kits well suited for multiplexing applications.

The large-scale antibody or 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 by a pre-packed spin column supplied with the kit and affords high antibody recovery (typically >85%). The entire labeling and purification procedure can be completed in as little as 90 minutes (approximately 15 mins hands-on time). Removal of free dye is essential for the determination of the degree of labeling (DOL). Everything needed to perform three conjugations is provided.

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Invitrogen Zenon labeling reagents provide a quick and easy-to-use system to noncovalently label human, mouse, and rabbit IgG antibodies with a variety of fluorophores. Prelabeled Zenon Fab fragments are designed to rapidly bind to the Fc portion of IgG antibodies, preserving antibody function. Zenon Fab-antibody conjugation occurs in less than 5 minutes, with only a small amount of starting material needed and no purification steps required.

The new Invitrogen Zenon Alexa Fluor Plus labeling reagents have been optimized to screen for antibody binding, giving a bright fluorescence-based indication of antibody binding. These reagents combine the rapid and scalable Zenon technology with the superior Alexa Fluor Plus fluorophores to provide a higher signal to noise ratio and to increase confidence in your screening results.

Want to learn more about screening for binding of therapeutic antibodies?
View our Scientific Poster for Antibody Labeling Reagents for Therapeutic Antibody Screening

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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).

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.

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.

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).

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.

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

*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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