cAMP- or cGMP-specific Phosphodiesterase Activity Kit

• Cat.No.: KITZ-064
• Product Overview: Cyclic nucleotide phosphodiesterases (PDEs) are involved in a myriad of cellular processes due to their ability to hydrolyze, and thus control, the levels of the second messenger signaling molecules cAMP and cGMP. PDEs have been implicated in a variety of diseases such as erectile disfunction, asthma, chronic obstructive pulmonary disease, learning disorders, depression and memory functions, neurodegenerative diseases, autoimmune disorders, heart failure and myocardial infarction. The cAMP- or cGMP-specific Phosphodiesterase Activity Kit is a luminescent, high-throughput screening (HTS) method for measuring cyclic nucleotide phosphodiesterase activity from purified sources. The kit is optimized to work with both cAMP- and cGMP-dependent phosphodiesterases. The total time required for the assay from start to finish is less than 1 hour after the PDE reaction is complete.
  cAMP- or cGMP-specific Phosphodiesterase Assay is compatible with 96-, 384- and 1536-well plate formats. The assay protocol begins by assembling a PDE reaction using cAMP or cGMP as a substrate at a final concentration of 1μM and 10μM, respectively. Following completion of the PDE reaction, PDE-Glo Termination Buffer containing the PDE inhibitor IBMX (3-isobutyl-1-methylxanthine, not supplied) is added to inhibit most PDEs; additional inhibitors may need to be added to this buffer. The PDE-Glo Detection Solution is added, then the Kinase-Glo Reagent is added. Luminescence is measured using a plate-reading luminometer. The light output is proportional to PDE activity in the reaction.

Specification

• Size: 1,000 assays
• Storage: Store the system at –10°C to –30°C.
• Shipping: Ice pack
• Kit Components: cAMP, 1mM: 100μl
  cGMP, 1mM: 500μl
  PDE-Glo Reaction Buffer, 5X: 1.2ml
  PDE-Glo Detection Buffer, 5X: 600μl
  PDE-Glo Termination Buffer, 5X: 600μl
  Protein Kinase A: 20μl
  Kinase-Glo Substrate: 1 bottle
  Kinase-Glo Buffer: 10ml
• Materials Required but Not Supplied: 1.100mM 3-isobutyl-1-methylxanthine (IBMX) in 100% DMSO
  2. additional PDE inhibitors, if desired
  3. white, opaque, polystyrene, nontreated, flat-bottom 96-, 384- or 1536-well plate. Do not use treated plates, black plates or clear plates.
  4. luminometer or charge-coupled device (CCD) capable of reading multiwell plates
  5. multichannel pipette or automated pipeting station
  6. purified active PDE enzyme(s)
  Note: To assay the PDE type I isoform add Ca2+ and calmodulin (CaM) to the PDE-Glo Reaction Buffer prior to assembling the PDE reaction.