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Assay Method Information |
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| | Enzyme Assay |
| Description: | PDEs specifically hydrolyze cAMP and/or cGMP and release the product AMP and/or GMP. The potency of PDE inhibition by test compounds is determined with a commercially available in vitro enzymatic assay (IMAP Fluorescence Polarization assay, Molecular Devices Corp.). Fluorescently labeled cAMP or cGMP is hydrolyzed by PDE preparations and in a second step, binding of labeled product to a large binding partner allows product detection by fluorescence polarization (FP) measurements.Stock solutions of the test compounds are made in DMSO and diluted in assay buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.1% BSA 0.05% NaN3, pH 7.2) to the desired concentrations. The solutions used in the assay contain the test compound in assay buffer with 2% DMSO. 5 μl of this pre-diluted test compound solution are mixed with 10 μl of substrate (FL-cAMP or FL-cGMP) at concentrations recommended by the manufacturer and with 5 μl of appropriately diluted PDE. 5 μl of reaction buffer with 2% DMSO are used for control reactions. The final concentration of DMSO in the assay is 0.5%, which does not significantly alter the PDE activity. After incubation for 90 minutes at room temperature, 60 μl of binding reagent are added as specified by the manufacturer. Binding is allowed to proceed for 30 minutes and fluorescence polarization is measured. |
| Affinity data for this assay | |
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