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Cyclic Nucleotide Phosphodiesterase assay kit

Unique colorimetric assay used to screen inhibitors and modulators of cyclic nucleotide phosphodiesterase activity.
 
BML-AK800-0001 96 wells 404.00 USD
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  • Unique, non-radioactive, colorimetric assay.
  • Used to screen inhibitors and modulators of cyclic nucleotide phosphodiesterase activity.
  • HTS format permitting rapid assays of large numbers of samples.
A BIOMOL GREEN™ Quantizyme Assay System. The Cyclic Nucleotide Phosphodiesterase (PDE) Assay Kit combines a special dual enzyme system with the BIOMOL GREEN™ Reagent for phosphate detection to create a unique, non-radioactive, colorimetric assay. 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 5’-nucleotide released is further cleaved into the nucleoside and phosphate by the enzyme 5’- nucleotidase. The phosphate released due to enzymatic cleavage is quantified using BIOMOL GREEN™ reagent in a modified Malachite Green assay. The kit includes Type I cyclic AMP phosphodiesterase (PDE) for validation purposes.

Product Specification

Alternative Name:PDE
 
Applications:Colorimetric detection, HTS
Activity assay
 
Use/Stability:For highest stability, store all components, except microplate (BML-KI101), at -70°C. Because its stability when frozen in solution is poor, the PDE enzyme (BML-KI456) is provided as 5 lyophilized aliquots. For highest activity, we recommend that each lyophilized aliquot be dissolved and used for one day’s experiments. The 5’-nucleotidase (BML-KI307) is used undiluted at 10 µl per well. It is stable for up to 6 freeze/thaw cycles (snap freezing with liquid N2 or dry ice/ethanol), but it may be desirable to make several aliquots if more numerous freeze/thaws are planned.
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Kit/Set Contains:PDE Enzyme (from bovine brain) (Prod. No. BML-KI456) (5 x 4 U; lyophilized solid, 4 U per vial. 1 U = 1 nmol 3’,5-cAMP to 5’-AMP per minute under the conditions of the linearity assay (30°C, pH 7.4, 200µM 3’,5’-cAMP (Prod. No. BML-KI180.) Storage: -70°C, avoid freeze/thaw cycles.
5’-Nucleotidase (from Crotalus atrox venom) (Prod. No. BML-KI307) (1ml; 5 kU/µl in 10mM TRIS-hydrochloric acid, pH 7.4, 125mM sodium chloride, 10% glycerol, 1mM magnesium chloride. One U will release 1 pmol phosphate per minute from 200µM 5’-AMP, 30°C in.a reaction buffer of 10mM TRIS-hydrochloric acid, pH 7.4, 0.2mM magnesium chloride). Storage: -70°C
3’,5’-cAMP Substrate (Prod. No. BML-KI180) (2ml; 1mM in assay buffer (10mM TRIS-hydrochloric acid, pH 7.4). Storage: -20 or -70°C
3’,5’-cGMP Substrate (Prod. No. BML-KI190) (2ml; 1mM in assay buffer (10mM TRIS-hydrochloric acid, pH 7.4). Storage: -20 or -70°C
PDE Assay Buffer (Prod. No. BML-KI181) (40ml (2 x 20ml); 10mM TRIS-hydrochloric acid, pH 7.4) Storage: -70°C
Biomol Green™ Reagent (20ml; liquid in screw-cap plastic bottle). Storage: 4°C, long-term at -70°C
5’-AMP Standard (Prod. No. BML-KI184) (1.0ml; 100µM in 10mM TRIS-hydrochloric acid, pH 7.4 buffer). Storage: -20 or -70°C
5’-GMP Standard (Prod. No. BML-KI182) (1.0ml; 100µM in 10mM TRIS-hydrochloric acid, pH 7.4 buffer). Storage: -20 or -70°C
Inhibitor (IBMX) (Prod. No. BML-PD140-9090) (200µl; 200µM in assay buffer (10mM TRIS-hydrochloric acid, pH 7.4)). Storage: -20 or -70°C
Desalting column and resin (Prod. No. BML-KI100) (1 column and 1 g of resin). Storage: room temperature, after rehydration store at 4°C
½ volume microplate (Prod. No. BML-KI101). Storage: Room temperature
 

Product Literature References

A novel systems pharmacology platform to dissect action mechanisms of traditional Chinese medicines for bovine viral diarrhea disease: C. Zheng, et al.; Eur. J. Pharm. Sci. 94, 33 (2016), Application(s): Experimental validation, Abstract;
Exposure to an Extremely-Low-Frequency Magnetic Field Stimulates Adrenal Steroidogenesis via Inhibition of Phosphodiesterase Activity in a Mouse Adrenal Cell Line: K. Kitaoka, et al.; Plos One 11, e0154167 (2016), Application(s): Phosphodiesterase (PDE) activity of desalted samples, Abstract; Full Text
Novel, primate-specific PDE10A isoform highlights gene expression complexity in human striatum with implications on the molecular pathology of bipolar disorder: C.M. MacMullen, et al.; Transl. Psychiatry 6, e742 (2016), Application(s): Measurement of PDE10A activity, Abstract;
Chromatographic fraction of Abrus precatorius leaf extract increases cGMP and nitric oxide concentration in female rats: I.H. Ogbuehi, et al.; Der Pharmacia Sinica 6, 29 (2015), Application(s): Determination of cyclic GMP levels in rats, Full Text
Constitutively Active Signaling by the G Protein βγ-Subunit Mediates Intrinsically Increased Phosphodiesterase-4 Activity in Human Asthmatic Airway Smooth Muscle Cells: A. Hu, et al.; PLoS One 10, e0118712 (2015), Application(s): Assay using HASM cell lysate, Abstract; Full Text
Cross-talk between PKA-Cβ and p65 mediates synergistic induction of PDE4B by roflumilast and NTHi: S. Susuki-Miyata, et al.; PNAS 112, E1800 (2015), Application(s): ELISA using whole cell extracts, Abstract; Full Text
Increased yield of high purity recombinant human brain natriuretic peptide by acid hydrolysis of short fusion partner in Escherichia coli: R. M. Kanumuri, et al.; Protein Expr. Purif. 111, 61 (2015), Application(s): Assay using human supernatant, Abstract;
Involvement of inducible nitric oxide synthase and dimethyl arginine dimethylaminohydrolase in Nω-Nitro-L-arginine methyl ester (L-NAME)-induced hypertension: M.D. Leo, et al.; Cardiovasc. Pathol. 24, 49 (2015), Abstract;
The cAMP phosphodiesterase Prune localizes to the mitochondrial matrix and promotes mtDNA replication by stabilizing TFAM: F. Zhang, et al.; EMBO Rep. 16, 520 (2015), Application(s): Assay, Abstract; Full Text
Hydrocortisone normalizes phosphodiesterase-5 activity in pulmonary artery smooth muscle cells from lambs with persistent pulmonary hypertension of the newborn: M. Perez, et al.; Pulm. Circ. 4, 71 (2014), Application(s): Used with fetal lamb pulmonary arterial smooth muscle cell (FPASMC) lysates, Abstract; Full Text
Synthesis of glycosides of resveratrol, pterostilbene, and piceatannol, and their anti-oxidant, anti-allergic, and neuroprotective activities: D. Sato, et al.; Biosci. Biotechnol. Biochem. 78, 1123 (2014), Abstract;
Cyclic stretch induces inducible nitric oxide synthase and soluble guanylate cyclase in pulmonary artery smooth muscle cells: M.R. Shah, et al.; Int. J. Mol. Sci. 14, 4334 (2013), Application(s): Use with cell lysates, Abstract; Full Text
Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: capturing transient ternary complexes by HDXMS: S. Krishnamurthy, et al.; Biochim. Biophys. Acta. 1834, 1215 (2013), Abstract;
Isolation of pulmonary artery smooth muscle cells from neonatal mice: K.J. Lee, et al.; J. Vis. Exp. 80, e50889 (2013), Abstract;
Persistent cAMP signaling by TSH receptors revealed by phosphodiesterase inhibition: E. Geras-Raaka, et al.; Thyroid. 23, 1484 (2013), Abstract;
Preserved cardiac function despite marked impairment of cAMP generation: M.H. Gao, et al.; PLoS One 8, e72151 (2013), Application(s): Use with cell lysates, Abstract; Full Text
β2-Agonists upregulate PDE4 mRNA but not protein or activity in human airway smooth muscle cells from asthmatic and nonasthmatic volunteers: K. Niimi, et al.; Am. J. Physiol. Lung Cell. Mol. Physiol. 302, L334 (2012), Application(s): Use with cell lysates, Abstract; Full Text
Caffeine increases mitochondrial function and blocks melatonin signaling to mitochondria in Alzheimer's mice and cells: N. Dragicevic, et al.; Neuropharmacology 63, 1368 (2012), Application(s): Use with cell lysates, Abstract;
Differential regulation of PDE5 expression in left and right ventricles of feline hypertrophy models: X. Shan, et al.; PLoS One 6, e19922 (2011), Application(s): Use with E.coli cell extracts and cardiac tissue lysates, Abstract; Full Text
Nitroarylhydroxymethylphosphonic acids as inhibitors of CD45: S. A. Beers et al.; Bioorg. Med. Chem. 5, 2203 (1997), Abstract;
Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) using synthetic phosphopeptides: K. W. Harder et al.; Biochem. J. 298 , 395 (1994), Abstract;

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