Targeting ENPP1 for Cancer Immunotherapy: Development of an HTS Method Using the Transcreener® AMP²/GMP² Assay

Transcreener AMP²/GMP² Assay (FP) Method

Ordering Information

Instrument Compatibility

Related Resources

Transcreener AMP²/GMP² Assay Kit - FP Readout

The Transcreener^® AMP²/GMP² Assay with FP readout measures AMP/GMP via antibodies selective to AMP/GMP and a far-red fluorescent tracer. AMP/GMP produced in the reaction competes with the tracer, decreasing fluorescent properties and providing a fluorescent readout. The universal detection method allows screening of virtually any AMP/GMP-producing enzyme in a high throughput format.

The assay produces an excellent signal at low substrate conversion for a broad range of initial ATP concentrations (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.

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

Fluorescence Polarization

Learn More About The AMP²/GMP² Assay

Transcreener AMP²/GMP² FP 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
Use with ATP/cAMP/cGMP concentrations ranging from 1 µM to 1,000 µM

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

Run your enzyme reaction, add Transcreener reagents, and read your plates. The AMP/GMP assay 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 FP Readouts Validated on Major Multimode Readers

Supplier	Instrument	FP Assays
	TriStar²S LB 942	In Review
	Tristar 5	In Review
	Mithras² LB 943	In Review

	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	Not Capable
	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	Not Capable
	SpectraMax^® Paradigm	Validated
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	Validated

	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
Δ mP ≥ 95 mP at 10% conversion of 10 μM ATP
Z' and Δ mP specifications to be met using Transcreener ADP² Assay reagents
Read time to achieve Z' and Δ mP 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 insure 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 optimal filter settings and maximal assay performance. We provide you with application notes with all 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
AMP²/GMP²Antibody	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. Sufficient antibody is included in the kit to complete 1,000 assays (Cat. #3015-1K) or 10,000 assays (Cat. #3015-10K).
AMP²/GMP² Alexa Fluor^® 633 Tracer	The final tracer concentration in the 20 µL reaction is 4 nM.
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/GMP² assays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphatases or nucleotidases, can produce background signal and reduce the assay window
Enzyme Buffer	User-supplied enzyme buffer components include enzyme, enzyme cofactors, substrates, and test compounds.
Assay Plates	Assay Plates Can Be Purchased Separately From BellBrook Labs. FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514.
Plate Reader	A multidetection microplate reader configured to measure FP of the Alexa Fluor^® 633 tracer 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 - FP 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
3015-1K	Transcreener AMP²/GMP² FP Assay (1,000 Assay, 384 Well)	$725.00	Add to Cart
3015-10K	Transcreener AMP²/GMP² FP Assay (10,000 Assay, 384 Well)	$4,099.00	Add to Cart
3015-100K	Transcreener AMP²/GMP² FP Assay (10 X 10,000 Assay, 384 Well)	$20,495.00	Add to Cart

Assay Plates for FP

Cat. No.	Product Description	Price	Add to Cart
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$55.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$60.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$479.00	Add to Cart