Kinetic Binding Assays (KON & KOFF)

Association: Cell or tissue homogenate containing the receptor of interest is incubated together with a single concentration of a labelled ligand with or without the presence of the non-labelled ligand (10 μM). The specific binding is measured at various times (0, 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min) in quadruplicate replicates at each tine point. Kinetic association constant (KON) will is calculated from this data.

Dissociation: Cell or tissue homogenate containing the receptor of interest is incubated together with a single concentration of a labelled ligand for 120 min. Then, dissociation is induced by adding 10 μM of non-labeled ligand and counted at different time points (0, 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min). Kinetic dissociation constant (KOFF) is calculated from this data.

The shape of the association and dissociation curves (monophasic vs biphasic) will is examined and the equilibrium dissociation constant (Kd) calculated from the kinetic constants.