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AMP2/GMP2 Assay for Screening Ligases, Synthetases and Phosphodiesterases

Ligase & Synthetase Assay - A Transcreener AMP2/GMP2 Assay Application

Transcreener AMP2/GMP2 Assay directly measures AMP/GMP produced by ligases & synthetases. These AMP/GMP measurements effectively determine the activity of the ligase & synthetase enzyme. The assay provides a powerful tool to screen compound libraries for ligase/synthetase modulators to help find new therapies for disease.

The kit comes complete with the detection reagents required to measure activity. Ligase/Synthetase enzyme is not included in the inhibitor screening asay kit. Please contact us for questions related to acquiring the enzyme.

How Does This Ligase & Synthetase Assay Work?

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

Transcreener Ligase & Synthetase Assay technology uses a simple but highly effective method that consists of an antibody selective to AMP/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 ligase & synthetase assay is available with FP and TR-FRET. It is 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 makes it extremely amiable to HTS.

Example Assay Target Applications

Enzyme Target
LigasesUb Ligases
SUMO Ligases
DNA ligases
RNA Ligases
SynthetasesAcetyl CoA Synthetase
Amino-Acyl tRNA Synthetases
NAD Synthetase

Direct Detection of AMP/GMP to Measure Ligase & Synthetase Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener Ligase & Synthetase Assay Schematic

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

Ligase & Synthetase TR-FRET Readout

Applications

  • Measure Enzymatic Activity of Ligases & Synthetases 
  • Screen Compound Libraries for Ligase/Synthetase Modulators
  • 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 minimize compound interference
  • A safe, non-radioactive assay technique
  • Wide substrate concentration range: 1 μM to 1000 μM

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

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

Transcreener Mix and Read Assay

Continuous Kinetic Mode Compatible

Transcreener Assays can be used in kinetic mode, allowing the assay to be used in applications not possible with endpoint only assay methods.

Measure Residence Times

Measure the residence times for inhibitors with a simple biochemical assay to characterize leads with the greatest potential to become drugs.

Streamlined Assay Development

Using the assay in kinetic mode reduces the number of experiments required to optimize conditions for a new enzyme.

Ligase & Synthetase 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 to accelerate your ligase/synthetase 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
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/iD5ValidatedClick 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

 

Edit
Component Notes
AMP2/GMP2 Antibody Antibody specific to the nucleotides AMP and GMP.
AMP2/GMP2 Tracer When displaced, changes in fluorescence can be read in an FP or TR-FRET readout, depending on the assay format.
Tris Solution Used to buffer the detection mixture.
AMP Used to create the cAMP/AMP standard curve.
GMP Used 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 Assay Kits

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

The Role of Ligases & Synthetases As Therapeutic Targets

The physiological role of ligases and synthetases is dependent on the respective family. Here, we explore roles of aminoacyl-tRNA synthetases as therapeutic targets.

Aminoacyl-tRNA synthetases ligate amino acids to tRNAs and translate the genetic code for protein synthesis. Because of their role in protein synthesis, these synthetases are needed for cell survival, and therefore are popular drug targets. Diseases they are targeted for include infectious diseases, cancers, and inflammatory diseases (Kim et al, 2020).  

The Transcreener AMP2/GMP2 Ligase & Synthetase Assay is an excellent tool for researchers examining the therapeutic effects of ligases and synthetases.

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