Homogeneous fluorescence assay for cyclic AMP

Combinatorial Chemistry & High Throughput Screening
Ewa HeydukTomasz Heyduk

Abstract

The objective of these studies was to develop a new homogeneous fluorescence assay for determining the concentrations of cAMP in biological samples. The assay is based on a novel general concept of using ligand-dependent sequence-specific DNA binding proteins as sensors for their respective ligands. CAP protein, a bacterial DNA binding protein whose DNA binding activity depends on cAMP, was used to develop the assay. In the presence of cAMP, DNA binding activity of CAP is greatly increased. Signaling of cAMP presence was achieved by detecting cAMP-dependent formation of CAP-DNA complex using a recently developed fluorescence assay for DNA binding proteins (Heyduk, T., and Heyduk, E. Nature Biotechnology 20,171-176, 2002). Both 96-well and 384-well black microplate formats of the assay were developed and used to detect cAMP in low nanomolar concentrations. The assay involves mixing of the sample with the assay solution containing all necessary components for cAMP determination followed by fluorescence intensity readout; no washing or reagent addition steps are necessary. Excellent reproducibility of fluorescence signal change as a function of cAMP concentration was observed. Experiments with HEK 293 cells stimulated with forskol...Continue Reading

Citations

Sep 27, 2007·PloS One·Daniel J KelleyAnita Bhattacharyya
Jan 16, 2010·Angewandte Chemie·Konstantinos LymperopoulosAchillefs N Kapanidis
Nov 10, 2011·Journal of Biomolecular Screening·Ling TianYie-Hwa Chang
Jul 13, 2006·Assay and Drug Development Technologies·Benjamin Dummitt, Yie-Hwa Chang
Apr 30, 2005·Analytical Chemistry·Ewa Heyduk, Tomasz Heyduk

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