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

Hi Arnaud,

Thanks so much for the response! I'm attempting to immobilize my ligand at a high enough level to see binding of a peptide analyte (Ligand MW=67000 Da, pI~4.6; analyte MW = 800-1000 Da, expected Kd = 1 uM - 500 nM). I know that the peptides I'm testing bind to the ligand in some capacity based on other assays, but I don't observe this via SPR (Biacore T200), so I reasoned that my low ligand immobilization must be the problem. I'm aiming for 7000-10000 RU based on my theoretical Rmax calculation of ~8000 for a response of ~100 RU. In the pictured immobilization, I used 50 ug/mL of my protein in 5 mM sodium acetate pH4.0 with a flow rate of 25 uL/min for 600s. I have also tried using 30 ug/mL of protein in 10 mM sodium acetate pH4.0 with essentially the same result (same slope during ligand injection). Both resulted in immobilization amounts of approximately 3900 RU (which again, did not show binding between the peptide analyte and the ligand).

I just repeated the immobilization at pH4.5 for 900s in 10 mM sodium acetate and observed a slope in alignment with your recommendation (~60 RU/s in the linear region of the injection curve), which is great, however I still see a similar drop between the response bound and the immobilized response (8400 RU vs 3600 RU, respectively). I plan to try pH 4.5 again with a longer contact time (maybe 1200s) to see if my immobilization improves. Do you have any other potential recommendations or alterations to my immobilization conditions?

Thanks again, it's always helpful getting a far more experienced opinion!
 

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

None of the listed additives contain a primary amine, so I don't reckon that's the issue. I'll try extending the contact time to see if that helps and if not, I'll go ahead and try a CM7 chip. If that doesn't work, perhaps I'll explore alternative immobilization methods - potentially biotinylating my ligand and using a SA surface may work.

Thanks again for the advice!