| Assay Method Information | |
| | In Vitro Detection of the Inhibitory Activity of Compounds Against PDE 3A Enzyme |
| Description: | Experimental objective: to determine the AMP/GMP expression based on fluorescence polarization, i.e., to trace binding of AMP/GMP to antibody so as to indicate enzyme activity.Reagents:Experimental buffer solution: 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.01% Brij 35, 1 mM Dithiothreitol (DTT), and 1% DMSO.Enzyme: recombinant human PDE3A (Gene accession number: NM_000921; amino acid 669-end) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag, with molecular weight being 84 kDa.Enzyme substrate: 1 μM cAMPDetection: Transcreener AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer.Procedure:1. The recombinant human PDE3A enzyme and enzyme substrate (1 μM cAMP) were each dissolved in newly-prepared experimental buffer solution;2. The PDE3A enzyme buffer solution was transferred into reaction wells;3. The compound which was dissolved in 100% DMSO was added to the reaction wells containing PDE3A enzyme buffer solution by acoustic technique (echo 550; millilambda range) and the mixture was incubated for 10 minutes at room temperature;4. The enzyme substrate buffer solution was added to the above reaction wells to initiate reaction;5. The resulting mixture was incubated at room temperature for 1 hour;6. The detection mixture (Transcreener AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer) was added to stop the reaction, and the resulting mixture was incubated for 90 minutes while slowly mixing. The measurement range of fluorescence polarization was Ex/Em=620/688.Data analysis: the fluorescence polarization signal was converted to nM based on AMP/GMP standard curve and the percentage enzyme activity relative to DMSO control calculated by Excel. GraphPad Prism was used for curve fitting (drawing medical icon). |
| Affinity data for this assay | |
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