Interaction changes with lipids

Hi all,
I am working with planar membrane chips and I am finding hard to have a good fitting.
In the first image there is a good 1 to 1 fitting of my protein, a Fab (arm of an antibody) (48KDa) attaching to a peptide in HBS.100ul/min. But when the peptide is embeded in a llipid membrane, the model is far from 1 to1. (fig 2). Also, if we look at the whole sensogram, it seems like the more protein I use, the more it gets attached to the membrane and won't dissociate.
So my questions are:

[/ul]Do you think, the curves are just bad, or there can be a bivalent interaction? lipid may affect the binding angle, and as usual, one to two fits well.
Looking at the whole sensogram, can I expect a normal one to one interaction?

I thank Arnoud for your support and encourage any other who works with lipids to write his experience too.
Regards

Miguel

Hi Miguel,

Please try to upload your figures ( www.sprpages.nl/kunena/new-forum/73-how-...n-image-to-your-post ).

Arnoud

Sorry, here are the figures

Hi Miguel,
The first fitting shows this buffer jump which is best visible between association and dissociation. This is a sign that the interaction is not entirely 1:1. Options to reduce non-ideal interaction are immobilizing less peptide and using pure analyte. The flow is already high. The second fitting is like the first although the dissociation is fitting less accurate. Also here, lowering the amount of peptide in the membrane can make the kinetics better. Do you use a reference like a membrane surface without peptide? This can help you to identify if the Fab is binding to the lipids of the membrane. Maybe you can alter the buffer (type, salt concentration and pH) to optimize the interaction. Most people stick to HBS but PBS or Tris is worth a try.

Kind regards
Arnoud

Yes, I use a reference channel with lipid alone. The figure of the total sensogram is after substracting the lipid channel component, that is why I don't understand why I have a fast and then a slow dissociation.
Maybe as you say, it is an artifact due to too much peptide.

Thanks for your advice.

Miguel

Hi Miguel,
Wondering if you are using a Nicoya system and liposome sensor.

Sonali