A BIOMOL GREEN
TM Quantizyme Assay System. The Cyclic Nucleotide Phosphodiesterase (PDE) Assay Kit combines a special dual enzyme system with the BIOMOL GREEN
TM 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 GREENTM reagent in a modified Malachite Green assay
1,2. The kit includes Type I cyclic AMP phosphodiesterase (PDE) for validation purposes.
Product Specification
| Quantity: | 96 assays |
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| 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 |
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| Long Term Storage: | -80°C |
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| 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. |
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| Background / Technical Information: | Please click here for the comprehensive product datasheet. |
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Product Literature References
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;
United States
