I initially did an FP assay with receptor (A) and a flourescent labelled ATP analog (B). For the first three times, i tried varying B conc for a fixed A concentration. I found increase in mP values with the highest increase occuring at the lowest ligand concentration. So if my A was fixed at 0.5 uM and B varied from 0.1 to 1 or 1.5 i got the highest increase for 0.1uM B. Still there was increase for all the conc of B but the rate of increase varied inversely with the conc of B. I then realized that to in order to get the Kd of the receptor A for ligand B, i need to reverse my experimenta strategy by varying the conc of A while keeping B fixed. I tried doing this with the following set up:

A varies from 0.05uM to 1.25uM while i have kept B either at 2.5uM or 0.25uM. In both the cases, all the (A,B) pairs show decline initially and then stabilize. I have been thawing and freezing my FL ATP analog and i am not sure if that has gone bad. However, accroding to my understanding, i should be getting increase FP signal with increase A concentration, because, if i increase the conc of E then more ATP will be converted to product ( say P-B) by the enzyme. And since, I did observe increasing mP values before when i varied B conc, my receptor is fine i guess ( i mean not aggregating or loss of function). Certain (A,B) pairs from both sets of experiments matched but for the first three times they showed increase while later they showed initia slight decrease and stablized. I do not understand this aberrant behaviour. Could anyone please explain?