Fig. 7.21 Kinetic MS binding study quantifying bound marker -dissociation experiment. Total binding (▼) of NO 711 (20 nM; incubation temperature 37 0C) to mGATl. GABA (10 mM) was added after 60 min preincubation to initiate dissociation (t = 0 min). Nonspecific binding (o) was determined as binding of NO 711 in the presence of 10 mM GABA. One representative example is shown. Data points represent each mean G SEM from triplicate values.
This yielded a k+1 of 0.0091 G 0.002 nM"1 min"1 for NO 711 binding to mGATl. Hence, the equilibrium dissociation constant calculated from kinetic MS binding experiments resulted in Kd = 11.7 G 2.5 nM. This is in good accord with Kd determined in MS saturation binding experiments and confirms the validity of the new setup.
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