Profiling PDEs with the Transcreener AMP²/GMP² Phosphodiesterase Assay

Transcreener AMP²/GMP² Assay (TR-FRET) Method

Ordering Information

Instrument Compatibility

Related Resources

Transcreener AMP²/GMP² Assay Kit - TR-FRET Readout

The Transcreener® AMP²/GMP² Assay kit with TR-FRET readout uses displacement of a tracer by AMP/GMP to measure enzymatic activity. This disrupts FRET from the antibody-bound Terbium chelate leading to a decrease in the TR-FRET signal. In addition to the time-resolved signal, the assay uses a far-red tracer to minimize interference from fluorescent compounds and light scattering.

The assay produces an excellent signal at low substrate conversions for a broad range of initial substrate concentrations (0.1-1,000 µM). Both reagents & signal are stable at room temperature for over 24 hours, providing outstanding flexibility for automated high-volume screening programs. The universal detection method allows screening of virtually any AMP/GMP-producing enzyme in a high throughput format.

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.

Direct Detection of AMP/GMP to Measure Enzymatic Activity

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

Learn More About The AMP²/GMP² Assay Kit

Transcreener AMP²/GMP² TR-FRET Assay Tech Manual

Applications

Measure enzymatic activity of AMP/GMP-producing enzymes
Screen compound libraries for enzyme modulators
Quantify inhibitor potency
Inhibitor selectivity profiling
Measure drug-target residence time

Features

Direct detection of unlabeled AMP/GMP
Robust Assay Z' > 0.7 under initial velocity conditions
Far-red fluorescent readouts minimize compound interference
A safe, non-radioactive method
Universal - use with virtually any AMP/GMP-producing enzyme

Easy-to-Use, Mix-&-Read, HTS Ready

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

One Assay... Hundreds of Targets

Along with PDEs the Phosphodiesterase Assay 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

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Far Red TR-FRET Readouts Validated on Major Multimode Readers

Supplier	Instrument	TR-FRET Assays
	TriStar²S LB 942	Validated
	Tristar 5	In Review
	Mithras² LB 943	Validated

	Cytation^TM 5	Validated
	Cytation^TM 3	Validated
	Cytation^TM 1	Validated
	Synergy^TM H1	Validated
	Synergy^TM 2/H4/4	Validated
	Synergy^TM HTX	Not Capable
	Synergy^TM Neo 2	Validated

	POLARstar^® Omega	Validated
	FLUOstar^® Omega	Validated
	PHERAstar^® FSX	Validated
	PHERAstar^® Plus/FS	Validated
	CLARIOstar^® /Plus	Validated
	VANTAstar^TM	Validated

	Sense	In Review

	Analyst^® GT/HT	Validated
	Gemini^® XPS/EM	Not Capable
	SpectraMax^® M2/M2e	Not Capable
	SpectraMax^® M5/M5e/FlexStation^® 3	Validated
	SpectraMax^® Paradigm	Contact Us
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	In Review

	EnVision^®/EnVision^® Xcite	Validated

	Infinite^® M1000/M1000Pro/Safire2^TM	Validated
	Infinite^® M200	Not Capable
	Infinite^® F500	Validated
	Infinite^® F200/Ultra^evolution	Contact Us
	Spark^TM 10M	Validated

Validation Criteria for Plate Readers

384-Well Format
Z’-Factor ≥ 0.7 at 10% conversion of 10 μM ATP
Z’ specifications to be met using Transcreener ADP² Assay reagents
Read time to achieve Z’ specifications ≤ 5 minutes

Look for these stickers on instruments that have successfully met the validation criteria.

The instrument validation program is a collaborative effort with the major suppliers of multimode readers to ensure that Transcreener assays perform optimally on your specific instrument. Scientists at BellBrook and/or the instrument vendor have tested Transcreener reagents with each instrument to determine the optimal filters and settings for maximal assay performance. We provide you with application notes with all of the important details, so you can be confident of success using Transcreener assays on the instrument of your choice.

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What's Included


Component	Notes
AMP/GMP HiLyte647 Tracer	The concentration of AMP/GMP HiLyte647 Tracer needed for an enzyme target depends upon the initial ATP/cAMP/cGMP concentration and buffer conditions in the enzyme reaction. Sufficient tracer is included in the kit to complete 1,000 assays (Part #3020-1K) or 10,000 assays (Part #3020-10K) at an ATP/cAMP/cGMP concentration up to 100 µM.
AMP²/GMP² Antibody-Terbium Conjugate	The final antibody concentration in the reaction is 4 nM in a 20 µL final reaction volume.
Tris Solution	Tris Solution is used to buffer the Detection Mixture.
AMP	AMP is used to create an ATP/AMP or cAMP/AMP standard curve
GMP	GMP is used to create a cGMP/GMP standard curve.

What You Will Need


Component	Notes
Enzyme	Transcreener AMP²/GMP²assays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphates and nucleotidases, can produce background signal and reduce the assay window.
Enzyme Buffer	User-supplied enzyme buffer components include enzyme, enzyme cofactors, substrate, and test compounds.
Assay Plates	Assay Plates Can Be Purchased Separately From BellBrook Labs. It is important to use assay plates that are entirely white with a nonbinding surface. We recommend Corning^® 384-well plates (Cat. #4513).
Plate Reader	A microplate reader configured to measure TR-FRET of the Tb:HiLyte647 donor:acceptor pair is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense a minimum volume of 2.5 µL into 384-well plates.
Ultrapure Water	Some 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.

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Transcreener AMP²/GMP² Assay Kits - TR-FRET Readout

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

Cat. No.	Product Description	Price	Add to Cart
3020-1K	Transcreener AMP²/GMP² TR-FRET Assay (1,000 Assay, 384 Well)	$789.00	Add to Cart
3020-10K	Transcreener AMP²/GMP² TR-FRET Assay (10,000 Assay, 384 Well)	$4,449.00	Add to Cart
3020-100K	Transcreener AMP²/GMP² TR-FRET Assay (10 X 10,000 Assay, 384 Well)	$22,245.00	Add to Cart

Assay Plates for TR-FRET

Cat. No.	Product Description	Price	Add to Cart
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$55.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$65.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$529.00	Add to Cart