Negative Response - Ligand Immobilization

Hello all, I have a question regarding negative response during ligand immobilization and would appreciate any suggestion.

I tried to immobilize my ligand after activation with NHS/EDC. At first injection at ~53 min I see a "normal" response, where an increase was observed upon injection. However when I tried injecting a second (~72 min) and third (~78 min) I saw negative responses. Is it because of non-specific interaction?

Any answer will be appreciated. Thank you.

Hi,
Can you tell us what buffers you used and what exactly you injected?
Also compare with www.sprpages.nl/immobilization/immobilization-procedures/amine

Arnoud

I used HEPES as running buffer and for coupling buffer I used sodium acetate at pH 0.5 lower than the pI of the ligand I was trying to immobilize.

Need some more information like concentration of ligand and are you using a carboxylated dextran surface?
And looking at the first ligand injection, it seems that there is immobilisation although the curve is not nice (too high ligand concentration?). When you saturate the surface the next ligand injections will not add any more.
Arnoud

I used CMD surface and the ligand concentration of each injection in 5 micromolar.