cAMP measurements are obtained using an ELISA assay (Harlow and Lane 1988). 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 that is described below. In outline, 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 calibrat e the assay.MATERIALSBuffers, Solutions, and ReagentsCellsAntibodiesSpecial EquipmentAdditional Equipment and ReagentsMETHOD REFERENCES Harlow E. and Lane D. 1988.Antibodies: A Laboratory Man ual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 579. Joseph E. and Guesdon J. L. 1982. Beta-galactosidase immunoassay for the measurement of cyclic AMP. Anal. Biochem.119: 335-340.Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the materi al set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult:Protein: Protein Interactions, Second Edition: A Molecular Cloning Manual, edited by Erica A. Golemis and Peter D. Adams, ? 2005 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 513.Copyright ? 2006 by Cold Spring Harbor Laboratory Press.All rights reserved. No part of these pages, either text or image may be used for any purpose other than personal use. Therefore, reproduction modification, storage in a retrieval system or retransmission, in any form or by any means, electronic, mechanical, or otherwise, for reasons other than personal use, is strictly prohibited without prior written permission.