Amine coupling low response

Hi SPR experts,

Recently I've been trying to immobilize a protein to a CM5 (Xantec and Cytiva) surface using EDC-NHS coupling. I've optimized the preconcentration steps, and observe significant (~25000 relative RU) accumulation of protein at the surface at pH 4.0 (The pI of this protein is ~4.6). However, following quenching with 1M ethanolamine, I observe a larger than expected drop in RU, from ~8500 RU immediately post-sample injection to ~3500 final immobilized RU. I know EDC-NHS reactions are less efficient at lower pHs, but I would expect that following analyte injection the relative response would be about as low as the final immobilized RU. Any thoughts/suggestions on what may be the cause of this problem and what I might do to solve it?

Thanks!

Hi and welcome,

What do you try to achieve?
The curve during ligand immobilization is way to steep - lower your ligand concentration or raise the pH. in order to control the ligand immobilization try to have a ligand immobilization slope between 25 - 100 RU/s.
In addition, a high pre-concentration does not mean a high covalent binding - as can be seen by the drop when ligand injection stops. Chemistry takes also time. Maybe read the SPRpages on this topic?

Any more information about the ligand - analyte pair and immobilisation conditions?

kind regards

Arnoud

Seems that something is preventing your protein to bind effeciently to the surface. Did you check the composition of the buffer of your ligand? It may not contain primary amines such as TRIS or Azide. In addition mayby other components are preventing coupling.
You can try to extend the activation time and look if this helps.
Last resourve could be using a high capacity sensor chip.

kind regards