Product namePDE Activity Assay Kit (Colorimetric)
Sample typeTissue, Inhibitor compounds
Assay typeEnzyme activity
Assay time0h 30m
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.
Storage instructionsPlease 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
Relevance3'5'-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
- (R,S)-Rolipram, Selective PDE4 inhibitor (ab120029)
- (R)-(-)-Rolipram, Selective PDE4 inhibitor (ab120031)
- Caffeine, Adenosine antagonist (ab120240)
- Dipyridamole, Non-selective phosphodiesterase (PDE) inhibitor (ab120451)
- RO 20-1724, Phosphodiesterase 4 (PDE4) inhibitor (ab141492)
- BRL-50481, Selective PDE7 inhibitor (ab141705)
- Sildenafil citrate, phosphodiesterase 5 (PDE5) inhibitor (ab141965)
- Amrinone, cAMP phosphodiesterase inhibitor (ab142259)
- Milrinone, phosphodiesterase 3 inhibitor (ab142322)
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.
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.
This product has been referenced in:
- Araiz C et al. Enhanced ß-adrenergic signalling underlies an age-dependent beneficial metabolic effect of PI3K p110a inactivation in adipose tissue. Nat Commun 10:1546 (2019). Read more (PubMed: 30948720) »
- Mukohda M et al. RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity. J Clin Invest 130:2318-2332 (2019). Read more (PubMed: 30896450) »