[7] Assay of Cyclic Nucleotides by Receptor Protein Binding Displacement

Receptor protein binding displacement assays for cyclic nucleotides are based on competition for protein binding sites between radioisotopically labeled nucleotide and the unlabeled material to be quantified. The binding proteins utilized are those that occur naturally and that interact with the appropriate cyclic nueleotide with high affinity. These procedures offer intrinsic advantages of simplicity of operation, high sensitivity, and high specificity. These features are particularly marked in the case of the assay for adenosine 3', 5'-cyclic monophosphate (cyclic AMP, cAMP) and have led to considerable utilization of this method. Binding activity may be simply followed through the purification procedure by incubation of small aliquots (5–25μl) of appropriate fractions with an excess of [³H] cAMP in 20 mM potassium phosphate, pH 6–7, for approximately 5 minutes at 30–37°C.