[The need for the correct choice of free ligand concentration in determining parameters of benzodiazepine binding]

In the studies of 3H-diazepam binding to the rat brain membranes it has been shown that insufficiently high concentrations of free ligand might lead to incorrect determination of Bmax. Thus, free ligand concentration in the range of 0.5 to 16 nM (the most often used ones) and low receptor-protein concentrations (0.08 to 0.12 mg/sample) were far from being saturating and therefore could not be applied for the analysis in Scatchard coordinates. In this case Bmax value would be considerably below the true Bmax value. It has been concluded that for the determination of Bmax of 3H-benzodiazepine binding the range of concentrations used should be at least 0.25 to 64 nM.