|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Bridge-It® PDE Assays |
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The Bridge-It® PDE Assay is a fluorescence-based, high-throughput screening (HTS) method for measuring cyclic nucleotide phosphodiesterase activity from purified source and determined 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). 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. Note: Definition of RF (% Quenching) is on page 8. 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.
Bridge- It® PDE Assay – PDE Activity Assay The following PDE activity assay is representative of those prepared using the Bridge-It® PDE assay. Note: Definition of RA (relative activity) is on page 8. Phosphodiesterase 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 6 pmole cAMP and 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 2 X assay solution was added and the signal was read after incubation at room temperature for 40 minutes.
Bridge- It® PDE Assay – IC50 Determination The following assay is representative of those prepared using the Bridge-It® PDE assay. Note: Definition of RA (relative activity) is on page 8. Phosphodiesterase 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, Cat#). 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 2 X 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. This product is protected by US Patent 6,544,746.
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
