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GALNT2 Assay - A Transcreener UDP2 Assay Application

Transcreener UDP² Assay directly measures UDP produced by GALNT2 (Polypeptide N-Acetylgalactosaminyltransferase 2). These UDP measurements allow researchers to effectively determine the activity of the GALNT2 enzyme. The assay provides a powerful tool to screen compound libraries for GALNT2 modulators to help find new therapies for disease. 

The kit comes complete with the detection reagents required to measure activity. GALNT2 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

How Does This GALNT2 Assay Work?

The Transcreener GALNT2 Assay determines GALTN2 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 GALNT2 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 GALNT2 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. 

Direct Detection of UDP to Measure GALNT2 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener UDP GALNT2 Assay Schematic

The workhorse. Used in many large screens. Best deck and signal stability.

Fluorescent Intensity (FI)

Transcreener UDP Glycosyltransferase Assay FI readout

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

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

Transcreener UDP Glycosyltransferase Assay TR-FRET Readout

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure Enzymatic Activity of GALNT2
  • Screen Compound Libraries for GALNT2 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 GALNT2 assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized GALNT2 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. Z' > 0.74. 

GALNT2 Assay Robust Data Z'
Z' GALNT2 Data Values

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

UDP-GalNAc Standard Curve
UDP-GalNAc Standard Curve Values

Detection of UDP Under GALNT2 Initial Velocity Conditions

GALNT2 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. 

GALNT2 Enzyme Titration

GALNT2 Assay Enzyme Titration

Linear Response

GALTN2 Linear Response Curve

Time Course - Detection of UDP Released by GALNT2

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.

GALTN2 Time Course Titration
GALNT2 Assay Time Course Standard Curve

GALNT2 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 GALNT2 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, FI & TR-FRET Readouts Validated on Major Multimode Readers

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
VANTAstar validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

What's Included

What You Will Need

The Role of GALNT2 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 GALNT2 Assay is an excellent tool for researchers examining the therapeutic effects of GALNT2.

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