Technical Notes

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

Issue link: https://resources.nanotempertech.com/i/1266323

Contents of this Issue

Navigation

Page 0 of 3

TECHNICAL NOTE Protein Labeling Site-specific covalent labeling of SNAP-tagged proteins for the measurement of binding affinities Introduction Site-specific fluorescent labeling of proteins is a powerful tool for the investigation of binding affinities by MST and TRIC 1 . 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 biochemistry 2–8 . 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 4 . 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 bond 9 . To utilize this tag for the fluorescent labeling of proteins, BG is conjugated to a fluorophore of interest, as illustrated in Figure 1. Ivana Bekić 1 , Matthias Molnar 1 , Oliver Beutel 2 , Edgar E. Boczek 2 , Sonia Joseph 2 , Matthäus Mittasch 2 , Amit Gupta 1 , Tanja Bartoschik 1 , Maria Knauer 1 1 NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich 2 Dewpoint Therapeutics, dewpointx.com

Articles in this issue

Links on this page

view archives of Technical Notes - Site-specific covalent labeling of SNAP-tagged proteins for the measurement of binding affinities