Application Notes

One-step, purification-free and site-specific labeling of polyhistidine-tagged proteins for MST

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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

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