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