Inhibitor Screening
To identify or confirm activity with the target.
BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
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 | |
|---|---|
| Ligases | Ub Ligases |
| SUMO Ligases | |
| DNA ligases | |
| RNA Ligases | |
| Synthetases | Acetyl CoA Synthetase |
| Amino-Acyl tRNA Synthetases | |
| NAD Synthetase |
Direct Detection of AMP/GMP to Measure Ligase & Synthetase Enzymatic Activity
Fluorescent Polarization (FP)
The workhorse. Used in many large screens. Best deck and signal stability.
Time-Resolved Förster Resonance Energy Transfer (TR-FRET)
For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.
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
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.
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
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.
Assay Services
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.
Lead Discovery Services Include:
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What’s Included
| 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
| Component | Notes |
|---|---|
| Enzyme | The assays are designed for use with purified enzymes. Use any enzyme that produces AMP, GMP, or CMP. |
| Enzyme Buffer | Enzyme buffer components optimized for your target. See the technical manual for potential interfering components. |
| ATP, cAMP, cGMP | Use the nucleotide required for your enzyme. It is possible to purchase some nucleotides direct from BellBrook Labs. |
| Assay Plates | Assay 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 Buffer | Some 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 Reader | A multi-detection microplate reader configured to measure the output of the AMP/GMP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes 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. |
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.
Resources
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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.