The SiteClick™ system allows 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, fluorescent dyes, metal-chelating compounds, and other small molecules like biotin—allowing multiplex analysis with antibodies from the same species.
Features of the SiteClick™ Antibody Labeling System
How the SiteClick™ Antibody Labeling System Works
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 1). 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 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.
|Figure 1. The SiteClick™ antibody labeling system. The first step in the SiteClick™ antibody labeling process involves removal of terminal galactose residues from the heavy chain N-linked glycans 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. In the third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-functionalized probe of choice (e.g., Alexa Fluor® 488 DIBO alkyne). The average degree of labeling is 3–3.5 labels per antibody.|
Custom and OEM Site-Selective Antibody Labeling Services
Do you have a “difficult-to-label” antibody? Does your antibody lose activity after labeling? Are you looking for a service that would custom-label your antibody in a site-selective manner? Our Custom Services department offers complete custom antibody labeling services, with many detection options that include Alexa Fluor® dyes, Qdot® probes, R-PE, biotin, and others. If you are interested in the GalT(Y289L) enzymatic antibody labeling kit alone (without the click reaction components), or in the custom synthesis of DIBO-modified reagents, please contact our Custom Services representatives for a quote by email at firstname.lastname@example.org.