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 GALNT3 Assay Work?
The Transcreener GALNT3 Assay determines GALNT3 enzyme activity by directly measuring the UDP formed by the enzyme using the Transcreener UDP Assay. By detecting UDP output, the assay provides a universal method to assess the activity of any UDP-producing enzyme in real-time.
Transcreener GALNT3 Assay technology uses a simple but highly effective method that consists of an antibody selective to UDP over UDP sugar donors and a far-red fluorescent tracer. UDP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The GALNT3 assay is available with FP, FI, 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 make it extremely amiable to HTS.
Applications
- Measure Enzymatic Activity of GALNT3
- Screen Compound Libraries for GALNT3 Modulators
- Quantify Inhibitor Potency
- Inhibitor Selectivity Profiling
- Measure Drug-Target Residence Time
Features
- Direct detection of unlabeled UDP
- Easy to use, homogenous, one-step format
- Robust Assay Z’ > 0.7 under initial velocity conditions
- Far-red fluorescent readouts minimize compound interference
- A safe, non-radioactive method
- Available in FP, FI, or TR-FRET readouts
Easy-to-Use, Mix-and-Read, HTS-Ready Assay
Run your enzyme reaction, add Transcreener reagents, and read your plates. The GALNT3 assay is compatible with 96, 384, and 1536-well formats.
Robust Assay Yields Quality Data
Z’ measurements using optimized GALNT3 reaction conditions indicate a robust assay. Robust data is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity.
UDP-GalNAc/UDP Standard Curve
The standard curve mimics an enzyme reaction (as UDP-sugar concentration decreases, UDP concentration increases). Z’ at 10% conversion (>0.85) shows excellent assay sensitivity under initial velocity conditions. The lower limit of detection is 100 nM UDP. Data shown here and for the following assays use the FI format. The UDP² Antibody-IRDye QC-1 concentration in the 10 µL reaction mixture is 1.049 µg/mL.
10 μM UDP/UDP-GalNAc Standard Curve
Detection of UDP Under GALNT3 Initial Velocity Conditions
GALNT3 was titrated in the presence and absence of Mucin 10-EA2 peptide (10 μM) and UDP-GalNAc (10 μM) for 60 minutes at room temperature. Data is converted to UDP formed using the UDP-GalNAc/UDP standard curve. The assay demonstrates a linear correlation under initial velocity conditions when raw data is converted to UDP using a standard curve.
GALNT3 Enzyme Titration
Linear Response
Time Course – Detection of UDP Released by GALNT3
Transcreener detection reagents were added to enzyme reaction prior to starting reactions by addition of UDP-GalNAc. Plates were then read at 30 minute intervals. Raw data in relative fluorescence units. Assay shows a linear correlation when converted to UDP formed.
GALNT3 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 and accelerate your GALNT3 program.
Lead Discovery Services Include:
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What’s Included In The Transcreener Kit
| Component | Notes |
|---|---|
| UDP² Antibody | Antibody specific to the nucleotide UDP. |
| UDP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| UDP | Used to create the UDP-sugar/UDP standard curve. |
Additional Materials You Will Need
| Component | Notes |
|---|---|
| GALNT3 Enzyme | We have successfully used recombinant human GALNT3 protein from R&D Systems #7174-GT-020. |
| Enzyme Buffer Components | Enzyme buffer components optimized for your targets. See the technical manual for potential interfering components. |
| EA2 Peptide Substrate | 10 μM of EA2 peptide from Anaspec #AS-63841 |
| UDP GalNAc Substrate | 10 μM of UDP GalNAc from Sigma-Aldrich #U5252. |
| 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 FI - 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 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the UDP 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 GALNT3 As a Therapeutic Target
Glycosyltransferases catalyze the transfer of sugar molecules to protein, lipid, and carbohydrate acceptors along with endogenous and xenobiotic small molecules. More than 200 human glycosyltransferases are known to exist and have diverse metabolic and regulatory roles. Of those, at least 20 distinct GALNTs (Polypeptide N-Acetylgalactosaminyltransferases) are found in the Golgi.
Since GALNTs catalyze the initial step of mucin-type O-glycosylation by transferring GalNac to Thr or Ser residues, these enzymes are targets for cancer interrogation. Multiple cancers directly link to GALNT overexpression and dysregulation. The Transcreener UDP GALNT3 Assay is an excellent tool for researchers examining the therapeutic effects of GALNT3.
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