Sometimes data does not contain sufficient information for an accurate kinetic analysis, such as with very rapid on/off-rates. In these cases, steady-state evaluations are more appropriate. The signal levels at equilibrium are plotted relative their concentration and a saturation curve is fitted to quantify the affinity. EC50, IC50 values and linear relationship can also be estimated using the same principle.
For fast on – fast off interactions, it can be more suitable to analyze signal levels than the binding rates. The affinity (KD) is the concentration corresponding to 50 % of the signal obtained at complete target saturation.