Abstract
In receptor binding assays with ultra-high-affinity radioligands, it is difficult, in practice, to adhere the golden rule that the receptor concentration in the assay should be substantially (at least 10-fold) lower than the dissociation constant (K(d)) of the radioligand and inhibition constant (K(i)) of compound. Especially for low specific activity radioligands (usually tritiated ligands of a couple of TBq/mmol), routinely applied in concentrations at around or below the K(d), the use of extremely small amounts of receptor protein per assay will result in low levels of bound radioactivity; the alternative use of larger assay volumes will make it difficult to apply 96-well filtration devices. For assessing the inhibition constant (K(i)) of competitive inhibitors under conditions violating the above golden rule, equations are available incorporating both [receptor] and [ligand] versus K(d); however, their application requires precise knowledge of [receptor] or initial bound/free [radioligand] ratio. In this study, we present the theoretical basis for determining the K(i) for a competitive inhibitor in a new protocol at high [protein] and high [radioligand] with the simple Cheng-Prusoff correction without the need to correct fo...Continue Reading
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