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Profiling PDEs with the Transcreener AMP²/GMP² Assay

Transcreener AMP2/GMP2 Phosphodiesterase Assay Kit

Transcreener AMP2/GMP2 Phosphodiesterase Assay directly measures AMP or GMP produced by a PDE reaction. This assay allow detection of any AMP or GMP producing enzyme (e.g. ligases, synthetases, phosphodiesterase) using any precursor substrate, including cAMP, cGMP, ATP, or NAD. It is the only activity assay method for direct detection of unlabeled AMP or GMP; i.e. without coupling enzymes.

The kit comes complete with the detection reagents required to measure activity. It does not include enzyme. Please contact us for questions related to acquiring the enzyme. This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

How Does The Assay Work?

The Transcreener AMP2/GMP2 Assay determines enzyme activity by directly measuring AMP or GMP formed by the enzyme. By detecting AMP or GMP, the assay provides a universal method to assess the activity of any AMP/GMP-producing enzyme in real-time. 

Transcreener technology uses a simple but highly effective method that consists of an antibody selective to AMP or GMP and a far-red fluorescent tracer. AMP/GMP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.

The assay is available with FP or TR-FRET in a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also make it extremely amiable to high throughput screening. 

Universal AMP/GMP Detection for Phosphodiesterases

Fluorescent Polarization (FP)

Transcreener AMP GMP FP Assay Schematic

The workhorse. Used in many large screens. Best deck and signal stability.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

Transcreener AMP GMP TR-FRET Assay Schematic

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®

Applications

  • Measure Enzymatic Activity of Phosphodiesterase
  • Screen Compound Libraries for Phosphodiesterase Inhibitors 
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled AMP or GMP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity reactions
  • Far-red fluorescent readouts minimizes compound interference
  • A safe, non-radioactive assay technique
  • Wide substrate concentration range: 1 μM to 1000 µM

One Assay … Hundreds of Targets

Along with PDEs the kit can be used with:

  • Ligases: Ub and SUMO Ligases, DNA and RNA Ligases
  • Synthetases: Acyl CoA Synthetase, Amino-Acyl tRNA Synthetases, NAD Synthetase
  • Phosphodiesterases: cAMP, cGMP
  • Sialyltransferases: (CMP detection)
  • Protein deAMPylase
  • Ectonucleotidases such as CD39, CD73, and ENPP1

Sensitive Assay with Large Window

Graph of Sensitivity Window of PDE41A

Measure the Potency of Inhibitors for a Variety of Targets

Potency of Inhibitors PDE41A and UbE1 Dose Response Curves

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.

Transcreener Easy-Mix-and Read Method

PDE Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your PDE program.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Far Red FP & TR-FRET Readouts Validated on Major Multimode Readers

SupplierInstrumentFP AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidated
Tristar 5In ReviewIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidated
CytationTM 5ValidatedValidated
CytationTM 3ValidatedValidated
CytationTM 1ValidatedValidated
SynergyTM H1ValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableNot Capable
SynergyTM Neo 2ValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableNot Capable
SpectraMax® M2/M2eNot CapableNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidated

What's Included

ComponentNotes
AMP2/GMP2 AntibodyAntibody specific to the nucleotides AMP and GMP. The concentration of AMP2/GMP2 antibody needed for an enzyme target is dependent upon the ATP/cAMP/cGMP concentration and buffer conditions in the enzyme reaction.
AMP2/GMP2 TracerWhen displaced, changes in fluorescence can be read in an FP or TR-FRET readout, depending on the assay format.
Tris SolutionUsed to buffer the detection mixture.
AMPUsed to create the cAMP/AMP standard curve.
GMPUsed to create the cGMP/GMP standard curve.

What You Will Need

ComponentNotes
EnzymeThe assays are designed for use with purified enzymes. Use any enzyme that produces AMP, GMP, or CMP.
Enzyme BufferEnzyme buffer components optimized for your target. See the technical manual for potential interfering components.
ATP, cAMP, cGMPUse the nucleotide required for your enzyme. It is possible to purchase some nucleotides direct from BellBrook Labs.
Assay PlatesAssay Plates Can Be Purchased Directly Through BellBrook Labs

FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514

TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513
Stop BufferSome PDEs and Ubiquitin Ligases have inhibited activity with Stop Buffer B (Cat. #2027-1 mL, Cat. #2032 10 mL) which quenches MgCl2.This allows the assay to be run in endpoint mode.
Plate ReaderA multi-detection microplate reader configured to measure the output of the AMP/GMP tracer is required.
Liquid Handling DevicesUse liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates.
Ultrapure WaterSome deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.

Transcreener AMP2/GMP2 Phosphodiesterase Assay Kits

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

PDE's As Enzyme Drug Targets

Phosphodiesterase's (PDE's) are enzymes that catalyze the hydrolysis of cAMP or cGMP to form AMP or GMP. cAMP and cGMP play important roles in a variety of biochemical processes. PDEs are currently classified into 11 different families. Each is classed based on a number of different factors including the distribution within tissue, and substrate specificity. Some hydrolyze cAMP, while others cGMP. A variety can degrade both cAMP and cGMP. 

PDEs have excellent promise in a variety of therapeutic areas including heart disease, dementia, infection, depression, and asthma. New inhibitors can provide better therapeutic potential and can limit off-target effects more effectively.

Cyclic nucleotide phosphodiesterases are proven drug targets. Most are a focus of inhibition due to their functional properties. Since PDEs degrade cAMP or cGMP, inhibitors can prolong the effects of the cyclic nucleotide in the signaling cascade within the tissues where the individual enzyme is most likely to reside.

Substrate Specificity

The Transcreener enables researchers to scour vast compound libraries for potent, yet selective inhibitors for unique PDEs. You can also complete a follow-up SAR to improve upon initial hits and increase the chance of success. 

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