Radioligands depletion

While the signal-to-noise ratio can be improved with increasing the amount of membrane used in binding studies, too much membrane can lead to depletion of radioligand with a concomitant introduction of errors in the estimates of ligand affinity. 

The equation for displacement of a radioligand [A ] by a nonradioactive ligand [B] can be rewritten in terms of one where it is not assumed that receptor binding does not deplete the amount of radioligand in the medium (no change in [A free]) ... 

The affinity constant of the test compound K can in turn be calculated from the IC50 value, the Kj of the marker and the concentration of M according to Cheng-Prusoff [see Eq. (3)] [22]. Since the concentration of free marker (M) is usually not determined in radioligand binding assays, it should be noted that the calculation of Ki according to Eq. (3) based on Mtot yields only reliable results if the depletion of the marker is negligible (i.e. <5% to 10% [7, 17, 21]). In competition experiments performed under typical conditions (Ttot Mtot this criterion is fulfilled. 

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