cAMP Assays
This protocol was adapted from “A Bacterial Two-Hybrid System Based on a cAMP Signaling Cascade,” Chapter 26, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.INTRODUCTION
The cAMP assay provides a quantitative determination of the efficiency of the functional complementation between pairs of hybrid proteins. cAMP measurements are obtained using ELISA. Commercial radio-immunoassays, or ELISA kits, to assay cAMP can be purchased from various manufacturers. In our laboratory, we use the homemade, less-expensive ELISA described here. This assay is based on the ability of soluble cAMP in bacterial extracts to compete with binding of an alkaline phosphatase (AP)-conjugated anti-cAMP antibody to immobilized cAMP. The surface-bound, immobilized anti-cAMP-AP is inversely correlated to the concentration of cAMP in the extract. Known concentrations of soluble cAMP are used to calibrate the assay.
