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Overview
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Product
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PDEase KitTM
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Description
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PDEase KitTM is designed to allow investigators to determine PDE4 activity
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Contents
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Incubation Buffer Reagent A
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5 mL
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Incubation Buffer Reagent B
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5 mL
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Incubation Buffer Reagent C
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5 mL
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PDE Substrate Buffer D
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10 mL
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PDE Substrate
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2X 200 µl
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Inhibitor Buffer
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50 mL
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Second Enzyme Source (10X)
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2X 250 µl
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Resin Slurry (10X)
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100 mL
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Control PDE4 Enzyme
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1x200 µl
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cAMP is a second messenger that is ubiquitously found in all mammalian cells. This messenger modulates the signal transduction of a variety of hormones, neurotransmitters, growth factors, cytokines and others ligand receptors. cAMP phosphodiesterase enzymes provide the sole pathway for hydrolyzing and truncating the cAMP signaling that is originated in response to receptor activation. The cAMP hydrolyzing activity is provided by a fairly large multi-enzyme family with distinct intracellular localization and regulatory properties. Among various PDE families (PDE1-PDE11), PDE4 has 4 genes (PDE4A, PDE4B, PDE4C, PDE4D) that are expressed in multi-splice variant forms.
The PDE4 activity measurement required a 2 step reaction, in the first reaction, cAMP is hydrolyzed by PDE activity in to non-cyclic form. This reaction is stopped by denaturing the PDE source or by addition of PDE4-selective inhibitor, followed by conversion of AMP to adenosine by the second enzymatic reaction to equilibrium. Since Adenosine is neutral, cAMP or AMP is separated by charge by ion exchange chromatography. In order to avoid background contribution of other PDEs, this PDE assay is carried out at substrate concentration optimized for PDE4. The PDE4 enzymatic activity is directly proportional to the adenosine formed in the first reaction.
This PDE4 activity measurement provides reproducible results within 2-3 hours. The kit provides enough reagents to process 50 or 100 samples in tandem along with internal controls. The kit reagents are stable for 6 months when stored at recommended conditions. The contents of this kit must be stored at different temperatures as indicated on the bottle labels and specification sheet.
PDEase Kit Protocol
SolObuffer Cell Lysis Kit
