Extraction and Assay of Cyclic Nucleotides

Cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) have between them been implicated in the mediation of many physiological phenomena. Over the last 30 years, a number of techniques have been employed to assess changes in the amounts of these two molecules. Many, however, suffered from lack of sensitivity and/or ease of performance. Since the early 1970s, two methods have been developed that to a large extent overcome these problems. The first is based on competitive binding to endogenous proteins (1 ). The second method is a competitive radioimmunoassay that, with minor modifications, can be used to detect levels of cyclic nucleotides down to 1 fmol/assay tube; this is discussed in detail below (2 ,3 ). The method is equally applicable to the measurement of all cyclic nucleotides; however, for clarity only that for cAMP is described.