APPLICATION NOTE
Protein Labeling
One-step, purification-free and site-specific labeling of polyhistidine-tagged
proteins for MST
Nuska Tschammer*, Stefanie Galinec*, Sebastian Weigert #, Yves Muller #, Changjiang You §,
Jacob Piehler §, Dennis Breitsprecher*
*NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich
# Division of Biotechnology, Department of Biology, University Erlangen-Nürnberg, Henkestr. 91, 91052 Erlangen
§ Division of Biophysics, Department of Biology, University Osnabrück, Barbarastr. 11, 49076 Osnabrück
Abstract
NanoTemper Technologies uses the popular polyhistidine-tag as target for an
unparalleled one step, purification-free labeling of proteins for MST experiments.
The labeling strategy employs a high affinity multivalent nitrilotriacetic acid (NTA)
derivative conjugated with the MST-optimized NT-647 dye (RED-tris-NTA). As the
hexahistidine tag (His6) provides binding sites for three NTA moieties, RED-tris-
NTA is perfectly suited for non-covalent, stable, highly selective labeling with a 1:1
stoichiometric ratio. The labeling can be performed with minute amounts of either
purified biomolecules or directly in the cell lysate.
Introduction
Nitrilotriacetic acid (NTA) and its derivatives have broad applications in the
manipulation of polyhistidine-tagged (His-tagged) proteins such as in immobilized
metal affinity chromatography (IMAC) (Hochuli, Dobeli et al., 1987, Ueda, Gout
et al., 2003) and surface immobilization (Gershon & Khilko, 1995, You & Piehler,
2014). Design of various multivalent NTA led to the discovery of tris-NTA, a
powerful tool to modify His-tagged proteins with low nanomolar affinity toward
hexahistidine tags (Huang, Hwang et al., 2009, Lata, Reichel et al., 2005). When
coupled to a fluorophore or biotin, tris-NTA efficiently labels His-tagged proteins
even in complex cellular systems (Kim, Jeyakumar et al., 2007, Lata, Gavutis et al.,
2006). This advanced tris-NTA moiety binds His-tagged proteins site-specifically