You are interested in experimentally determining the ligand binding

affinity of a cell-surface receptor with Kd = 10 pM and association rate

constant kon = 105 M1s1. You incubate nine separate cell suspensions

with the following concentrations of fluorescently labeled ligand: 0.1,

0.3, 1.0, 3.0, 10.0, 30.0, 100.0, 300.0, and 1,000.0 pM. Simulate the

binding isotherm (binding curve) you would obtain if you measured

cell-bound fluorescence at the following times: 15 minutes, 1 hour, 4

hours, 16 hours, and 64 hours. You may assume pseudo-first-order

kinetics. Curve-fit each of these five isotherms to obtain an estimate

of Kd. Is it necessary for every sample to reach equilibrium in order to

obtain an accurate Kd estimate?