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Quantification of cAMP and cGMP analogs in intact cells: pitfalls in enzyme immunoassays for cyclic nucleotides.


ABSTRACT: Immunoassays are routinely used as research tools to measure intracellular cAMP and cGMP concentrations. Ideally, this application requires antibodies with high sensitivity and specificity. The present work evaluates the cross-reactivity of commercially available cyclic nucleotide analogs with two non-radioactive and one radioactive cAMP and cGMP immunoassay. Most of the tested cyclic nucleotide analogs showed low degree competition with the antibodies; however, with Rp-cAMPS, 8-Br-cGMP and 8-pCPT-cGMP, a strong cross-reactivity with the corresponding cAMP and cGMP, respectively, immunoassays was observed. The determined EIA-binding constants enabled the measurement of the intracellular cyclic nucleotide concentrations and revealed a time- and lipophilicity-dependent cell membrane permeability of the compounds in the range of 10-30% of the extracellular applied concentration, thus allowing a more accurate prediction of the intracellular analog levels in a given experiment.

SUBMITTER: Werner K 

PROVIDER: S-EPMC3145891 | BioStudies | 2011-01-01T00:00:00Z

REPOSITORIES: biostudies

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