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Site-specific covalent labeling of SNAP-tagged proteins for the measurement of binding affinities

Site-specific fluorescent labeling of proteins is a powerful tool for the investigation of binding affinities by MST and TRIC1. This labeling strategy preserves the biochemical and physicochemical properties of proteins due to its targeted approach and its one fluorescent label per protein molecule stoichiometry. Additionally, it prevents the interference of fluorophores with ligand binding, making it an attractive alternative to conventional, covalent labeling approaches.

In addition to the His-tag, the SNAP-Tag® is another protein tag that is already used for site-specific labeling for numerous applications in biochemistry2–8. This 20 kDa protein tag is commercially available in various expression vectors, allowing its fusion to any protein of interest. Since this tag can be added to the N- or C-terminal end of proteins, it has typically no effects on the protein functionality.

The SNAP-Tag® is a modified form of the DNA repair enzyme, human O6-alkylguanine- DNA alkyltransferase (hAGT), which specifically reacts with O6-Benzylguanine (BG) derivatives to form an irreversible covalent thioether bond9. To utilize this tag for the fluorescent labeling of proteins, BG is conjugated to a fluorophore of interest, as illustrated in Figure 1.

SNAP-Tag® labeling is an ideal tool for stable, covalent, and highly selective labeling of purified biomolecules with minimum impact on the protein’s biochemical and physicochemical properties.

With the intention to exploit the use of SNAP-Tag® for site-specific protein labeling in binding affinity assays, BG was conjugated with the TRIC-optimized RED 2nd Generation dye to form the RED-SNAP-Tag® 2nd Generation, included in the SNAP-Tag® Labeling Kit RED 2nd Generation (Cat# MO-LO19). To demonstrate the applicability of this labeling strategy, affinity analysis of a SNAP-tagged RNA-binding protein towards RNA molecules was performed.

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