Application Notes

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

Issue link:

Contents of this Issue


Page 1 of 7

2 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. situ non-covalent, stable, highly selective labeling of biomolecules carrying the His6 tag in a stoichiometric ratio of 1:1. With the intention to exploit the use of His-tags for site-specific protein labeling in MST experiments, the tris-NTA scaffold was conjugated with the MST-optimized NT-647 dye (RED-tris-NTA). To demonstrate the versatility and superiority of the novel NanoTemper Technologies RED-tris-NTA dye, an array of MST experiments was performed on different proteins, which were either purified or present in crude cell lysate. Results and Discussion Labeling The versatility, robustness and specificity of the unique RED-tris-NTA dye were tested with a peptide as well as proteins in different labeling buffers, crude cell lysate and in the presence of various potential interfering compounds and additives. The affinity of the dye towards a His6 peptide, a cancer-associated isocitrate dehydro-genase point-mutant (His6-IDH R132H) and mitogen-activated protein kinase 14 (p38α) was determined by MST (Figure 2). The His6 peptide, His6-IDH R132H and His6 p38α were titrated against 10 nM of RED-tris-NTA in PBS-T buffer. The observed K d values were 1.3 ± 0.2 nM for the His6 peptide, 0.6 ± 0.3 nM for IDH R132H and 2.4 ± 1.1 nM for p38α. The minor deviation in the K d between different His-tags are most likely due differences in the accessibility and the local electrostatic potential within the respective proteins, or due to slight inaccuracies in the determination of the protein stock concentrations. in stoichiometrically defined and stable manner. Moreover, because of its small size, binding of tris-NTA has minimum effect on biochemical and physicochemical properties of the protein. Figure 1: Chemical structure of the NT-647 conjugated tris-NTA (RED-tris-NTA) and schematic representation of RED-tris-NTA – protein interaction. The conjugate is shown loaded with nickel(II) ions as used for the site-specific labeling of His-tagged proteins. Two remaining coordination sites of the NTA- complexed nickel(II) ions are occupied by general ligands X, and can interact with His moieties of His-tags. The basic mechanisms of the interaction between the NTA-based label and histidines is governed by the intrinsic property of the imidazole group present in the histidine to chelate nickel(II) nitrilotriacetate (Ni(II)-NTA) (Terpe, 2003). The hexahistidine tag (His6) provides six binding sites precisely matching the three Ni(II) ions complex of the NTA moieties (Figure 1) (Lata et al., 2005). Tris-NTA is thus perfectly suited for in

Articles in this issue

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