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. , 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. 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.
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