Thiol - antibody immobilization

To whom it may concern
I am using Biacore and i am trying to immobilize thiol
polymer on gold surface. I have difference responce in
channels often. When the difference before immobilize the
polymer is 2000 i can immobilize the antibody with out
proplem, but if the difference is 4000 all the Ab is removed
with the first regeneration. Do you think that if much
polymer is immobilized on surface this doesnt allow the Ab
to bind strongly with polymer?? My final regeneration will
be Ab-Antigen. I use HCl 0,01M as regeneration solution with
SDS 0,1%. Do you think is the appropriate regeneration
solution?
Thanks.
Best Regards
Dimitris Kyprianou

I understand you are immobilizing the antibody by adsorption to the polymer coated surface, right? One possible explanation to the vehabior you observe is that when you have a lower density of polymer the antibody binds to the gold surface as well. This binding can be stronger that the binding with the polymer and would explain that the antibody is not leaving the surface after the first regeneration. At higher polymer density you would avoid in a greater extent the binding of your ligand to the gold. I would recommend you to run a control experiment, flowing the antibody over a plain gold surface to detect possible adsorption of the ligand to the gold.