Dear Alice,

to find the origin of the binding problem, I would start with simple checks of the preparation. First of all, does your Histag work in the IP? It could be buried if the protein is native. You should determine the concentration of the eluted protein and also check the eluate on an SDS-PAGE for purity / copurified proteins.

Second, precipitation on the column is theoretically possible if your protein is not stable, so you could again compare pre-/post desalting concentrations and PAGE results.

Finally, Ni2+ binding is dependent on ionic strength and pH, so re-check the buffers for the IP beads and the SPR chips according to the manufacturer's recommendations.

I hope this gives you a starting point.

Best,

Marianne

Marianne Musinszki

Thank you very much for your suggestions.

The Histag works perfectly on IP, it's actually pretty sticky on the Ni-NTA beads. I did both Comassie and SDS page for my protein (before and after desalting) and the concentration it's quite high (around 2-1.5 mg/ml depending on the prep). It's not amazingly pure, but should be enough for binding to the chip.

I might try to change the ph of the buffer I use during the experiment, I've been using HSB-P buffer ph 7.4, it could be I get better results with ph 7 or 8.

Thank you again!

Alice

Hello Alice Nardin,

the concentration should be definitely enough.

Are there differences between the "IP-NTA" and the "chip-NTA"?

If you are loading the Ni2+ on the chip yourself, you may check the protocol used, maybe the efficiency is not as high as you expect.

I assume you don't use EDTA in your buffers.

I never tried it myself, but there also is the possibility to do a covalent linking using the amine groups after immobilization in the desired orientation via Ni-NTA.

Maybe it can even be done alternately to saturate more binding sites on the chip.

Good luck!