Does anyone have experience in receiving negative binding signals when analyzing small molecules (500-1000Da) binding to immobilized proteins (100kDa). It would be interesting to hear about possible explanations and systems where this was seen. The sensorgrams are analyzable and show saturation binding yielding good results so the system seems to work anyhow.
As an explanation a conformational change in the protein dependent on binding could compensate the positive binding signal of the analyte binding, but who knows....
thanx for the reply and for the very good and infomative homepage. It's very useful to have a forum to ask questions about Biacore experiments to a world wide community.
The negative sensorgrams are seen before and after referencing (double referencing). I have measure different analytes with the same result using this specific target. reversing the system has not been done yet. that would be a possibility but a difficult one because of the nature of the analyte. Our approach is to try to measure a heavier analyte to compensate for a possible conformational change or a matrix deformation yielding negative resonses.
The obtained KD values do correspond to a equilibrium assay (so there must be some kind of truth (correlation) in the biacore assay). Strangly the Rmax (or Rmin looking at the untreated sensorgrams) is dependent on how much target is immobilized.
First your last remarks. Good: KD's should be verified with different set ups so your are on the right way.
Is it possible to do ITC (isothermal calorimetrie) or CD (circular diochroism). The latter is best equipped to demonstrate conformational change.
Rmax should differ with the amount of ligand immobilized because Rmax is the response by the injected analyte. Rmax depends on the Molecular weights of the ligand and analyte and the amount (binding places) of the immobilized ligand. Don't confuse Rmax with Req. Rmax is only approached by > 100x KD.
These analyte bindings are they fast (ka >10e6, kd < 10-2) or slow?
the analytes shows fast association (10e5) and dissociation (10e-1). Until now, I have measured three different analytes and always seen the same negative sensorgrams and the correlation to the other assay.
ITC is planned for this system. I hope that I'll be able to confirm the results using it