No binding

Dear SPR pages,
I have been trying to do Kinetics on this sample set: a mAb with an Analyte that has Mw of ~25kDa. I am seeing No Binding at all. I am using a Capture method with ProteinA immobilized on a CM5 chip. The mAb binds really well on the Protein A surface, but No binding seen for the Analyte. Completely flat. I did find information on the Analyte sample, it has a His-tag on it. Is this the issue? How can I do SPR on protein samples that has a His-tag on it?
Thanks.

HI,

It is possible that a HIS-taq is interfering with the binding to the antibody but I would be supprised to see that. You can immobilize an anti HIS antibody to capture your HIS-tagged-protein.
Maybe first coat the protein in an ELISA plate and confirm that the mAb is recognising your protein before trying more SPR.

Kind regards
Arnoud

ok, will check on the Elisa. Also, is it also possible to capture the His-tag protein using an NTA chip? Saw that Biacore sells these NTA chip, activated by injecting a solution of Ni2+ ions first then capturing the histidine-tagged proteins.

Yes, the NTA-chip is especially designed for the capture of HIS-tagged proteins.
The advantage is that you can strip the HIS-tagged protein from the surface and reload it with a different one.
kind regards
Arnoud