Ligand non-specifically binding to NTA chip

Hello,

I'm currently trying to develop an assay for interaction between His-tagged protein and one specific ligand using T200 and NTA chip. The protein immobilises easily (~1500 RUs) and is pretty much stable. Buffer only injections look as expected as well. The problem is with the ligand.

It is a natural product that has 4 basic amines and steroid like structure. In pH 7.5 the amines are presumably totally protonated. After injection, I get higher binding to the reference surface than to the Protein surface (2-1 is negative) the higher the concentration of the ligand becomes.

A few questions:

Is it possible this positively charged ligand (base) is binding non-specifically to the reference surface due to opposite charges? + and -? also the ligand having steroid like structure makes it quite hydrophobic as well.

If that's true, would it possible to somehow neutralise the charge on the reference surface of NTA chip? Maybe another random protein on reference surface could maybe decrease this random binding? Something positively charged? Lyzosyme? BSA? (BSA has steroid binding capabilities though...) Any other thoughts?

Thanks for your answer!

Hi,

Basic proteins tend to bind to the dextran matrix of the sensor chip. Since you use the NTA-sensor chip to capture the ligand you have to deal with the dextran matrix. You can try to play a little with the pH, e.g. go to 8.5 in steps of 0.5 unit and see if the non-specific interaction is less. Adding a bit more NaCl can mask some charges and lower non-specific interaction.
Shielding the matrix with another non-related protein is an option but you should test this on a protein-by-protein basis.

Other options are to immobilize the basic protein on the sensor chip and use the HIS-tagged protein as analyte or immobilize the HIS-tagged protein on a flat surface (no dextran, low carboxylation).

Kind regards
Arnoud