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Enzyme Immunoassay for Studying Intracellular Levels of cAMP

emoved by a wash step. The reaction is completed by the addition of the colorimetric substrate, 3,3,5,5-tetramethylbenzidine/hydrogen peroxide (TMB/H2O2). The addition of TMB/H2O2 to each well results in a blue-green product background, and the reaction is stopped by the addition of 1 M phosphoric acid. A bright yellow color results, and absorbance of peroxidase-labeled cAMP that is bound to the cAMP-specific antibody is determined using a spectrophoto- metric plate reader at 450 nm. Since the amount of cAMP-horseradish peroxidase bound to rabbit anti-cAMP antibody is inversely proportional to cAMP in the sample (or standard), a high absorbance value corresponds to low levels of cAMP present in the sample. The EIA cAMP Kit includes cAMP standards for calculating standard curves. As an option for increased sensitivity (figure 2), standards and prepared samples can be acetylated by treatment with acetic anhydride in the presence of triethylamine.1 Picomolar detection is possible for acetylated cAMP, whereas nonacetylated cAMP levels can generally be assayed in the nanomolar range. The EIA cAMP Kit includes all the re- agents and protocols for quantifying cAMP levels in both nonacetylated and acetylated preparations.

AC Toxin for Increased Intracellular cAMP Levels

Stratagene is offering adenylate cyclase (AC) toxin, a useful reagent for increasing intracellular cAMP levels in cultured cells. AC toxin can be used to study cAMP-dependent protein kinase activation, the key regulatory event in signal transduction pathways involving cAMP. Modulators currently available for examining the effects of cAMP on intracellular signaling include
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