I am trying to regenerate an NTA chip. My ligand is HIS-tagged streptavidin and the analyte I bind to this is biotintylated anti-IgG. I am able to regenerate the chip when only the ligand is bound by using 200 mM Imidazole. However, when I bind the anti-IgG to the HIS-tagged streptavidin, I am not able to regenerate the NTA chip. I have tried the following regeneration solutions:
1. Imidazole 200 mM and 1M
2. HCl 10 mM and 20 mM
3. Glycine-HCl 10 mM
4. EDTA 20 mM and 350 mM
5. 0.5% SDS
Does anyone have any suggestions on regeneration solutions that might work in this case? Or any explanations as to why this is the case?
The interaction between the HIS-tagged streptavidin and the Ni-NTA moiety on the chip is a 1:1 binding. This interaction is easily broken with imidazole leaving the Ni-NTA moiety intact. When using EDTA the Ni is stripped from the NTA moiety and loading the NTA with Ni is necessary to let it work again. This is relatively straight forward.
When binding the biotinylated IgG to the streptavidin, a more complex interaction occurs. For instance, the streptavidin has four binding spots and the IgG will also have several biotins bound to it. I can imagine that this multivalent interaction is more resistant to regeneration solutions. If the packing is dense it can even cover the entire sensor chip.
Finding the best regeneration solution (if possible) will be something of trial and error. You have covered the usual solutions already. I can suggest looking at
and try the cocktail regeneration as suggested by K. Andersson.
1. Andersson, K., Areskoug, D. and Hardenborg, E.; Exploring buffer space for molecular interactions. J.Mol.Recognit. (12) 5: 310-315; 1999.
2. Andersson, K., Hamalainen, M. and Malmqvist, M.; Identification and optimization of regeneration conditions for affinity- based biosensor assays. A multivariate cocktail approach. Analytical Chemistry (71) 13: 2475-2481; 1999.
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