The NTA-sensor chips use the property that Ni2+ can chelate a 6 x HIS-sequence. This makes the NTA-sensor chip easy and versatile to use.
Rich, Rebecca L. et al Biacore analysis with stabilized G-protein-coupled receptors. Analytical Biochemistry 409: 267-272; (2011). Goto reference
Biacore analysis with stabilized G-protein-coupled receptors. This publication shows an improved method for tethering His-tagged receptors on NTA (carboxymethylated dextran preimmobilized with nitrilotriacetic acid) chips to yield stable, high-capacity, high-activity surfaces as well as a novel approach to regenerate receptor binding sites.
Knecht, S et al Oligohis-tags: mechanisms of binding to Ni2+-NTA surfaces. J.Mol.Recognit. 22: 270-279; (2009). Goto reference
Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni2+ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C- or N-terminal histidine (His) tag. In this study the binding mechanism of His-tags to Ni2+-NTA was investigated. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS).
Nieba, L. et al BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Analytical Biochemistry 252: 217-228; (1997).
The histidine (His) tag can be routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag. This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here.
Lata, S. and J. Piehler Stable and Functional Immobilization of Histidine-Tagged Proteins via Multivalent Chelator Headgroups on a Molecular Poly(ethylene glycol) Brush. Analytical Chemistry 77: 1096-1105; (2005). Goto reference
A generic approach for immobilizing oligohistidine-tagged proteins with high stability and homogeneous functionality onto glass-type surfaces is presented. Multivalent chelator heads (MCH) carrying two and three nitrilotriacetic acid (NTA) moieties were coupled with controlled surface concentration to glass surfaces premodified with an ultrathin two-dimensional polymer brush of a bifunctional poly(ethylene glycol). Low roughness and lateral homogeneity of these surfaces were confirmed by AFM and fluorescence microscopy, respectively. Protein immobilization and interactions at these interfaces were studied by label-free and fluorescence detection. Oligohistidine-tagged proteins bound specifically to NTA loaded with nickel(II) ions and could be eluted with imidazole. More than 90% of the immobilized protein preserved its activity. In contrast to mono-NTA, immobilized multivalent chelator heads bound oligohistidine-tagged proteins stoichiometrically and with high stability, even at very low chelator surface concentrations. Thus, an excess of the metal chelator sites was not necessary, and excessive binding sites could be quantitatively blocked with an indifferent protein. As a consequence, increased functional stability of the immobilized protein and a substantial reduction in nonspecific adsorption were achieved.