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

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

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. 

Mix-and-Read-GAP-Assay

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

Far Red FP Readouts Validated on Major Multimode Readers

SupplierInstrumentFP Assays
TriStar2S LB 942In Review
Tristar 5In Review
Mithras2 LB 943In Review
CytationTM 5Validated
CytationTM 3Validated
CytationTM 1Validated
SynergyTM H1Validated
SynergyTM 2/H4/4Click Me ArrowValidated
SynergyTM HTXNot Capable
SynergyTM Neo 2Validated
POLARstar® OmegaClick Me ArrowValidated
FLUOstar® OmegaNot Capable
PHERAstar® FSXClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidated
SenseIn Review
Analyst® GT/HTClick Me ArrowValidated
Gemini® XPS/EMNot Capable
SpectraMax® M2/M2eNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not Capable
SpectraMax® ParadigmClick Me ArrowValidated
SpectraMax® iD3/iD5Click Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidated
Infinite® M200Not Capable
Infinite® F500Click Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidated

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. 

What's Included

What You Will Need

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