PDE6C Assay Kit

• Background:

  Phosphodiesterases (PDEs) play an important role in the dynamic regulation of the second messengers cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate) signaling, by hydrolyzing them. The PDE superfamily is composed of 11 families, with PDE4,7 and 8 being cAMP-specific hydrolases, and thus regulating positive and negative responses to it. PDE6C, also known as cGMP-inhibited phosphodiesterase, is involved in light detection and cone phototransduction. PDE6C encodes the alpha-prime subunit of cone-specific phosphodiesterase, an enzyme found exclusively in the light-detecting (photoreceptor) cells called cones. Cone cells are found in a specialized tissue at the back of the eye known as the retina, and mutations in PDE6c are associated with eye diseases including Cone Dystrophy 4 and Achromatopsia. Continuing to understand PDE6C roles and the development of newer specific therapeutic tools will open new treatment avenues in PDE-related disorders.

• Description:

  The PDE6C Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well or 384-well assay kit designed for the screening and profiling of PDE6C (Phosphodiesterase 6C) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE6C, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 or 400 reactions.Figure 1: Illustration of the PDE6C Assay Kit principle.The assay uses a fluorescein-labeled cyclic guanosine monophosphate (cGMP-FAM for PDE6C), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE6C catalyzes the hydrolysis of the phosphodiester bond in the cyclic nucleotide and frees the phosphate group. In a second step the free phosphate group is recognized by a specific phosphate-binding nanobead (Binding Agent) leading to the formation of a large complex, with restricted movement (high FP) . FP is proportional to PDE6C activity.This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) and equipped with the required parts to read the FP signal. For more information FP technology, visit our Tech Note:FP, assay principles and applications.Note:As of July 2024, this protocol has been re-optimized for performance. Previous versions of this kit are available upon requestNeed us to run inhibitor screens or profile your compounds against PDE6C? Check out ourPhosphodiesterase Screening Services.

• UniProt:

  P51160

• Applications:

  Study enzyme kinetics and screen small molecule inhibitors for drug discovery in high throughput screening (HTS) applications.

• Format:

  96 reactionsCatalog #NameAmountStorage60062PDE6C, GST-Tag*˃1 µg-80°C60201FAM-Cyclic-3′, 5′-GMP**2 x 1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete) 25 ml-20°C60390PDE Binding Agent100 µl4°C60392Binding Agent Diluent (cGMP) 10 ml4°C0.5 M DTT200 µl-20°C79685Low binding, black 96-well plate1Room Temp* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.**FAM-Cyclic-3′, 5′-GMP is provided as a powder. Each vial will need to be resuspended in 400 µl of Complete PDE Assay Buffer before use.384 reactionsCatalog #NameAmountStorage60062PDE6C, GST-Tag*˃1 µg-80°C60201FAM-Cyclic-3′, 5′-GMP**3 x 1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete) 25 ml-20°C60390PDE Binding Agent250 µl4°C60392Binding Agent Diluent (cGMP) 25 ml4°C0.5 M DTT200 µl-20°C79961Low binding, black 384-well plate1Room Temp* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.**FAM-Cyclic-3′, 5′-GMP is provided as a powder. Each vial will need to be resuspended in 800 µl of Complete PDE Assay Buffer before use.

• Shipping Conditions:

  -80°C

• Storage Conditions:

  This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed

• Notes:

  Troubleshooting GuideVisitbpsbioscience.com/assay-kits-faqfor detailed troubleshooting instructions. For further questions, email[email protected]

• Contraindications:

  The final concentration of DMSO in the assay should not exceed 1%.Fluorescent compounds that have λex=470 nm and detection at λem=528 nm can interfere with the readouts.It is recommended that the compound alone is tested to determine any potential interference of the compound on the assay results.