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
