BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip.

  title={BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip.},
  author={Lars Nieba and S E Nieba-Axmann and Anders V. Persson and Markku D. H{\"a}m{\"a}l{\"a}inen and F Edebratt and Annasara Hansson and Jonas Lidholm and Karin Magnusson and Anneli Karlsson and Andreas Pl{\"u}ckthun},
  journal={Analytical biochemistry},
  volume={252 2},
While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, 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… 

Figures and Tables from this paper

Oligohis‐tags: mechanisms of binding to Ni2+‐NTA surfaces

The binding mechanism of His‐tags to Ni2+‐NTA was investigated and it was demonstrated that two His separated by either one or four residues are the preferred binding motifs within hexahis tag, and elongation of these referred motifs decreased affinity.

Long Term Sensibility of the Ni-Co Protein Chip

A Ni-Co protein chip whose intermediate metal surface could capture the recombinant protein successfully and provide specific binding with His-tagged biotin and His- tagged Uricase is developed based on the optimization approach of Taguchi method.

High-affinity chelator thiols for switchable and oriented immobilization of histidine-tagged proteins: a generic platform for protein chip technologies.

This new platform for switchable and oriented immobilization should assist proteome-wide wide analyses of protein-protein interactions as well as structural and single-molecule studies.

An integrated, peptide-based approach to site-specific protein immobilization for detection of biomolecular interactions.

An integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes and is compatible with high-throughput procedures is developed.

Development of a new and simple method for the detection of histidine-tagged proteins.

Covalent affixation of histidine-tagged proteins tethered onto Ni-nitrilotriacetic acid sensors for enhanced surface plasmon resonance detection of small molecule drugs and kinetic studies of antibody/antigen interactions.

The Ni2+-histidine (His) chelation yields a more uniform and predicable orientation of immobilized protein molecules than an amine-coupling reaction in surface plasmon resonance (SPR) analyses.

Multivalent chelators for spatially and temporally controlled protein functionalization

The application of multivalent chelators (MCH) for high-affinity yet reversible recognition of oligohistidine (His)-tagged proteins and the key principles and features of MCH–His-tag interactions are introduced.



A self-assembled monolayer for the binding and study of histidine-tagged proteins by surface plasmon resonance.

Surface plasmon resonance studies showed that His-tagged proteins adsorbed on the NTA-SAM retained a greater ability to participate in binding interactions with proteins in solution than protein immobilized in a thin dextran gel layer by covalent coupling.

Detection and quantitation of hexa-histidine-tagged recombinant proteins on western blots and by a surface plasmon resonance biosensor technique.

The synthesis and use of biotinyl-nitrilotriacetic acid is described which, in combination with streptavidin-horseradish peroxidase, allows for the detection of hexa-histidine-tagged recombinant proteins on Western blots.

Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions.

Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.

Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent

We describe a general purification method for recombinant proteins based upon the selective interaction between a poly-histidine peptide, which is fused to the protein of interest, and a novel metal

Interpretation of deviations from pseudo-first-order kinetic behavior in the characterization of ligand binding by biosensor technology.

Results suggest that the most common source of deviations from the pseudo-first-order kinetic approximation of BIAcore kinetic data is likely to be heterogeneity of the immobilized ligand sites.

Competition BIAcore for measuring true affinities: large differences from values determined from binding kinetics.

It is shown that binding constants in solution can be reproduced well by using on-rate determinations of antibody preincubated with antigen, and the conditions under which such an approach is valid are derived.