Dear RG researchers,

I have been wondering is there any way to calculate the Ki value of an agonist in the [35S]GTPgammaS assay? It only gives the EC50 value, but is it possible to obtain the Kd value of the radioligand in this assay to later calculate the Ki of the stimulating agonist from the Cheng-Prusoff equation? I thought about using increasing concentrations of unlabeled GTPgammaS against a single concentration of the labeled GTPgammaS with 8-OH-DPAT as the stimulating ligand (the target is the 5-HT1A receptor) to calculate the IC50 and then use the simplified Cheng-Prusoff equation: Kd = Ki = IC50 − [radioligand concentration] used for homologous competitive binding experiments to calculate the Kd. Is this in any way legit? Or running the competition binding assay is the only reliable option to establish the Ki? I would be grateful for any suggestions.