regeneration conditions

Hello,

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?

Thanks a lot
Blanca

Hi Blanca,
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.

More regeneration hints can be found at www.sprpages.nl/kinetics/regeneration.html

Arnoud