Immobilization on NTA-Chips

Hello Together!

I have recently started a project where I want to use SPR to study protein-carbohydrate interactions. However, I have never worked with SPR before which makes me a total beginner.

I would like to immobilize a His(6)-tag protein. In the literature I found out that NTA-chips are commonly used for this purpose. So I ordered a Xantec NiHC-200M (NTA-derivatized polycarboxylate hydrogel). While reading up on the methodology, I now have a few questions.

First of all some information:
- Device: Biacore T200
- protein approx. 30 kDa with N-terminal His(6) tag
- analyte approx. 20 - 25 kDa

Now my questions:
Can this kind of immobilization (activation with nickel + injecting liagnd) be permanent? The protein I want to immobilize is very expensive and therefore I don't want to re-inject it for every experiment (over a time of 3 months). However, I keep reading in the literature that after immobilization and analysis of the ligand-analyte-interaction, you regenerate the surface of the chip (e.g. with 350 mM EDTA). But with this, my surface is going to be totally regenerated, so my immobilized protein is gone?
Can I skip this regeneration so that my protein continues to remain on the surface? And if so, is it storable this way? Or do I run the risk that my analyte will also remain on the surface?

One more question about this: I also came across a paper where His(6) proteins were covalently immobilized on an NTA chip using EDC and NHS. Does anyone perhaps have experience with this or a protocol?

I am sorry for all that questions.
I would be very happy if someone could help me.

Best regards,
Joe S.

Hi Joe,
The sensor chip from Xantec is ok to use to capture a His-tagged protein. The term capture gives it away: this is not a permanent bonding between sensor surface and ligand. Normal kinetics apply to the NTA-6xHIS interaction.
Depending on the strength / stability of the interaction you can use the surface several times to inject analyte over it but this is not recommended since each run you lose some ligand and regeneration is difficult (in case the analyte does not dissociate completely). Therefore this kind of surface is single analyte injection only. The surface can be mildly regenerated with imidazole to strip off the ligand (the nickel is kept on the sensor surface) or with EDTA in case you have to load new nickel. In either case you have to reload the ligand. This is a drawback in relation to ligand consumption but when you ligand is not easily regenerable a NTA-chip with ligand capture is the best choice.
I would not recommend storage of a NTA-chip with ligand. It is better to strip and load with nickel before storage.

The protocol to capture the ligand and use NHS/EDC is useful to make covalent bonding between sensor surface and ligand providing that the surface contains free –COOH groups to activate and make amine bonds. You should ask Xantec for this. In addition, keep in mind that there will also covalent bound formed between ligand molecules possibly lowering the functional activity of the surface.

Also compare look at the references at www.sprpages.nl/sensor-chips-intro/biacore-sensor-chips/nta .

Kind regards
Arnoud