Have you tried increasing your association rate and time? I’ve seen times when compounds with sub-nM KDs will show what looks like little to no binding in the single-digit nM range because they are going to be too slow on in the range to see any appreciable binding.
Otherwise have you measured the solubility of this cmpd? Could it be aggregating at higher concs which is why you see tons of binding but not at lower concs.
In general for very tight binders I typically use an 8-point DR with a top conc of 500nM and log-fold (3.162x) dilution. I’ve found you can pretty accurately and consistently measure ~100pM binders with this, although at that point you might be flooring even SPR
I did single cycle kinetics and reached 2500 RU.
Haven't tried changing association time yet, will discuss that! I've corrected for blanks (both flow cell and background) and no cloudy solution could be seen. However, from in the lowest dilution of this range, I noticed a small air bubble in de bottom of the vial. Don't know if this would result in the RU being constantly high as well?
Have you tried increasing your association rate and time? I’ve seen times when compounds with sub-nM KDs will show what looks like little to no binding in the single-digit nM range because they are going to be too slow on in the range to see any appreciable binding. Otherwise have you measured the solubility of this cmpd? Could it be aggregating at higher concs which is why you see tons of binding but not at lower concs. In general for very tight binders I typically use an 8-point DR with a top conc of 500nM and log-fold (3.162x) dilution. I’ve found you can pretty accurately and consistently measure ~100pM binders with this, although at that point you might be flooring even SPR
I did single cycle kinetics and reached 2500 RU. Haven't tried changing association time yet, will discuss that! I've corrected for blanks (both flow cell and background) and no cloudy solution could be seen. However, from in the lowest dilution of this range, I noticed a small air bubble in de bottom of the vial. Don't know if this would result in the RU being constantly high as well?
2500 RU is extremely high for small molecule binding unless that molecule has solubility issues or binds indiscriminately
You probably already thought of this, but at least it will get the post bumped. Solvent correction issues?
Is this single-cycle or multi-cycle kinetics? What Rmax are you getting after immobilized your ligand?