I've an anti-His antibody immobilized (with amine coupling chemistry) on a CM5 sensor chip (7000 RUs aprox. in both Fc2, Fc1). I passed an His-tagged protein and got 360 RUs aprox on Fc2. After that, I wanted to test a regeneration strategy but the 10mM acidic regeneration solutions (pH from 3 to 1.5) fail to remove my ligand or, at least, I don't see a change in the baseline at all. In the past, 10mM glicine pH 1.5 has worked for me but I don't have too much experience. Is it common that the ligand stays on the antibody at that low pH? What regeneration condition would you try next?
The Glycine solutions give sometimes strange results, like higher baseline. I do not know why.
What you can try is to use a different regeneration solution. Try a short pulse of 10 mM HCl for a low pH.