NTA Chips for SPR

NTA sensor chips can be successfully used for immobilization of molecules carrying more then two his-tags, as shown by Nieba et al. [1], e.g. with oligomeric proteins. Sometimes immobilization can even succeed with two his-tags per molecule, while affinity of singly his-tagged proteins to the NiNTA complex is insufficient to immobilize such proteins.

Also, Nieba et al.[1] studied several other parameters affecting level of ligand immobilization (starting point conditions listed in parentheses, but they may need to be re-optimized for a particular system):

• conditions for NTA surface activation with Ni²⁺: [NiCl₂], [NaCl] and pH of activation buffer (good starting points: 0.3-4 min pulse 300μM NiCl₂, pH 6.4-8.4, 0-0.5M NaCl)
• effects of pH, [NaCl] and detergent (tween) concentration in the ligand immobilization buffer
• effect of Na₂EDTA (0-300 mM) in the running and ligand buffers (50 μM EDTA does not negatively impact ligand immobilization, recommended to scavange free metal ions in solution competing for His-tag binding)
• concentration of his-tagged ligand (<1 μM, needs to be determined experimentally - too much ligand prevents multidentate binding and some his-tagged protein will be leaching out)

Surface regeneration conditions were optimized as well.

References

1. Nieba L, Nieba-Axmann SE, Persson A, Hämäläinen M, Edebratt F, Hansson A, Lidholm J, Magnusson K, Karlsson AF, and Plückthun A. BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem. 1997 Oct 15;252(2):217-28. DOI:10.1006/abio.1997.2326 |