Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions

Chunxia Zhao, Lance M. Hellman, Xin Zhan, Willis S. Bowman, Sidney W. Whiteheart, Michael G. Fried

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


The hexahistidine (His6)/nickel(II)-nitrilotriacetic acid (Ni2+-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nanogold to His6 proteins. Here we explore several applications of the NTA derivative, (Ni2+-NTA)2-Cy3. This molecule binds our two model His6 proteins, N-ethylmaleimide sensitive factor (NSF) and O6-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K ∼ 1.5 × 106 M-1) and no effect on their activity. Its high specificity makes (Ni2+-NTA)2-Cy3 ideal for detecting His6 proteins in complex mixtures of other proteins, allowing (Ni2+-NTA)2-Cy3 to be used as a probe in crude cell extracts and as a His6-specific gel stain. (Ni2+-NTA)2-Cy3 binding is reversible in 10 mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, but in their absence it exchanges slowly (kexchange ∼ 5 × 10-6 s-1 with 0.2 μM labeled protein in the presence of 1 μM His6 peptide). Labeling with (Ni2+-NTA)2-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions that prevent direct detection of protein (e.g., high ADP absorbance). In addition, fluorescence resonance energy transfer (FRET) between (Ni2+-NTA)2-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances.

Original languageEnglish
Pages (from-to)237-245
Number of pages9
JournalAnalytical Biochemistry
Issue number2
StatePublished - Apr 15 2010


  • Analytical ultracentrifugation
  • Bis-NTA-Cy3

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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