Bridge-It® cAMP-Phosphodiesterase (PDE) Assay Kit, 384-well format

PDE PD1007-384

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Description

Bridge-It® Phosphodiesterase (PDE) Assay Kit, 384-well format

The Bridge-It® Phosphodiesterase (PDE) Assay is a fluorescence-based, high-throughput screening (HTS) method for measuring cyclic nucleotide phosphodiesterase activity from a purified source and determining the IC 50 of their inhibitors. This assay is based on the format of Bridge-It® cAMP all in one Fluorescence Assay (Mediomics, Cat# 122938).

 

Applications:

  • PDE-related drug discovery projects

  • Research on heart, lungs (i.e. COPD), platelet function, and inflammatory mechanisms

  • Research on anxiety disorders or neurodegenerative diseases

 

Features:

  • Easy: Mix test sample or standard with the Assay solution and incubate at ~25°C

  • Fast: Read fluorescent signal after 30 minutes of incubation

  • Flexible: Assay is adaptable to both low- and high-throughput screening formats

 

Pricing: The 100-well kit is a one-time trial purchase. For bulk pricing options, please contact us. For any other questions or comments, please do not hesitate to contact us. We are always happy to help!


 Background Information on cAMP, PDE, and the Bridge-It Fluorescence Assay

Adenosine-3’,5’-cyclic monophosphate (cAMP)

Adenosine-3’,5’-cyclic monophosphate (cAMP) is an important second messenger which is involved in the modulation of numerous biological processes. The measurement of cAMP is especially important in new drug discovery since cAMP levels are closely related to the activity of one of the major targets for new drug discovery – the G protein-coupled receptors (GPCR). Mediomics fluorescence cAMP assay method for determining the concentrations of cAMP is based on the assay platform design described above. Basically CAP, a bacterial DNA binding protein whose DNA binding activity depends on the presence of cAMP, is used as a highly specific biosensor for measuring cAMP levels. As PDE hydrolyzes cAMP to AMP, the concentration of cAMP decreases, which results in less of the CAP-DNA complex formation and more free “half-site” duplexes with probes (unquenched). The increase in the fluorescence signal is proportional to the PDE activity.

 

Bridge- It® PDE Assay – cAMP Standard Curves

The following cAMP standard curve is representative of those prepared using the Bridge-It® PDE assay. It shows a standard curve prepared in Buffer B in a 384-well plate with the fluorescence signal read after 30 minutes, 1 hour, and 2 hours after addition of 2X Assay Solution.

camp_pde1

Bridge- It® PDE Assay – PDE Activity Assay

The following PDE activity assay is representative of those prepared using the Bridge-It® PDE assay. The relative activity was measured in samples with the indicated amount of PDE from bovine brain (Sigma, St. Louis, MO, Cat# P9529). The reaction was incubated at room temperature for 30 minutes and stopped by addition of 0.5 ul stop solution. The 2X assay solution was added and the signal was read after incubation at room temperature for 40 minutes.

camp_pde2

Bridge- It® PDE Assay – IC50 Determination

The following assay is representative of those prepared using the Bridge-It® PDE assay. The relative activity was measured in samples with 0.8 mU PDE from bovine brain (Sigma, St. Louis, MO, Cat# P9529) and the indicated amount of IBMX (Sigma, St. Louis, MO).

camp_pde3

 

Note: Definition of RA (relative activity) is on page 8 of the protocol. The PDE assay was performed in a standard 384-well plate in a final 10 ul reaction in 1 x PDE reaction buffer (25 mM Tris, pH 7.4, 100 mM NaCl, BSA 0.1 mg/ml, CaCl2 0.03 mM, Calmodulin (Sigma, St. Louis, MO, Cat# P1431) 10 U/ml), with 0.8 mU PDE from bovine brain (Sigma, St. Louis, MO, Cat# P9529) and the indicated amount of IBMX (Sigma, St. Louis, MO). The enzyme and IBMX were mixed and pre-incubated at room temperature for 5 minutes. 6 pmole cAMP was added, and the reactions were incubated for 30 minutes at room temperature and stopped by addition of 0.5 ul stop solution. The 2X assay solution was added and the signal was read after incubation at room temperature for 40 minutes.

RESEARCH USE:For research use only, not for use in diagnostic procedures.

 

For information on the assay principle please see the Bridge-It Assay Technology platform page or download the protocol. This product is protected by patents and pending patents.


Selected Customer Publications using Mediomics’ Bridge-It® cAMP-PDE Fluorescence Assay Kit:

1. Tan W, Thomas P. Activation of the Pi3k/Akt pathway and modulation of phosphodiesterase activity via membrane progestin receptor-alpha (mPRalpha) regulate progestin-initiated sperm hypermotility in Atlantic croaker. Biol Reprod. 2014 May;90(5):105

— “Phosphodiesterase activity was measured using the Bridge-It cAMP PDE Assay kit (Mediomics, St. Louis, MO).”

2. Zhu H, Guariglia S, Li W, Brancho D, Wang ZV, Scherer PE, Chow CW. Role of extracellular signal-regulated kinase 5 in adipocyte signaling. J Biol Chem. 2014 Feb 28;289(9):6311-22.

— “Phosphodiesterase activity was determined by using the Bridge-It cAMP-PDE assay kits from Mediomics.”

3. Garciandia A, Suarez T. The NMRA/NMRAL1 homologue PadA modulates the expression of extracellular cAMP relay genes during aggregation in Dictyostelium discoideum. Dev Biol. 2013 Sep 15;381(2):411-22.

— “Phosphodiesterase activity was measured in freshly obtained conditioned media, from 1×10 7 cells/mL starved for 6.5 h (plated at 2×10 6 cells/cm2), using Bridge-It cAMP_PDE Assay (Mediomics, LLC, IL, USA).”

 

 

Catalog #: PD1007-100 , PD1007-384

Protocol : 

MSDS :