PDE Activity Assay Kit (Colorimetric) (ab139460)
Key features and details
- Assay type: Enzyme activity
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 30 min
- Sample type: Inhibitor compounds, Tissue
Overview
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Product name
PDE Activity Assay Kit (Colorimetric) -
Detection method
Colorimetric -
Sample type
Tissue, Inhibitor compounds -
Assay type
Enzyme activity -
Assay time
0h 30m -
Product overview
PDE Activity Assay Kit (Colorimetric) ab139460 combines a special dual enzyme system with Green Assay Reagent for phosphate detection to create a unique, non-radioactive, colorimetric assay to detect phosphodiesterase (PDE) activity. This HTS-friendly, mix and read system may be used to screen inhibitors and modulators of cyclic nucleotide phosphodiesterase activity. 96-well microplate format permits rapid assays of large numbers of samples. The basis for the assay is the cleavage of cAMP or cGMP by a cyclic nucleotide phosphodiesterase.
The kit includes a Type I PDE as positive control and a non-specific PDE inhibitor, 2-isobutyl-1-methylzanthine (IBMX) as test control for inhibitor screening.
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Platform
Microplate reader
Properties
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Storage instructions
Please refer to protocols. -
Components 1 x 96 tests 3',5'-cAMP Substrate 1 x 2ml 3',5'-cGMP Substrate 1 x 2ml 5' AMP Standard 1 x 1ml 5'-GMP Standard 1 x 1ml 5'-Nucleotidase (from Crotalus atrox venom) 1 x 1ml 96-well Clear Microplate (1/2 Volume) 1 unit Desalting Column 1 unit Desalting Resin 1 x 1g Green Assay Reagent 1 x 20ml PDE Assay Buffer 1 x 40ml PDE Enzyme (from bovine brain) 5 vials PDE Inhibitor 1 x 200µl -
Research areas
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Relevance
3'5'-cyclic nucleotide phosphodiesterases are a family of phosphodiesterases. Generally, these enzymes hydrolyze a nucleoside 3’,5’-cyclic phosphate to a nucleoside 5’-phosphate. Some examples of nucleoside 3’,5’-cyclic phosphate include: 3',5'-cyclic AMP, 3',5'-cyclic dAMP, 3',5'-cyclic IMP, 3',5'-cyclic GMP, 3',5'-cyclic CMP
Images
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Standard Curve for 5’AMP
Duplicate wells of 5’-AMP dilutions were prepared. Phosphate was released from 5’-AMP by incubation with 5’-nucleotidase (50 kU/well, 30°C, 30 min.) and the reaction terminated by addition of Green Assay Reagent (100 µL/well). After 30 min., the phosphate-dependent color reaction was measured by reading OD620nm in a microplate-reading spectrophotometer.
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Time Course of cAMP Hydrolysis by PDE, Inhibition by IBMX.PDE enzyme (20 mU/well) was incubated with cAMP (200 μM) and 5’-nucleotidase (50 kU/well) with or without the inhibitor IBMX (40 μM) at 30°C for the indicated times. Reactions were terminated by addition of 100 µL of Green Assay Reagent and OD620nm read 30 min. later. A cAMP standard curve may be used to convert OD620nm data to nmol of 5’-AMP.