Discovery of GALNT3 Inhibitors Using a Universal Glycosyltransferase Assay and Orthogonal Pooled Screening

Transcreener UDP Assay (FP) Method

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Instrument Compatibility

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Transcreener UDP² Assay Kit - FP Readout

The Transcreener^® UDP² Assay with FP readout measures UDP via antibodies selective to UDP and a far-red fluorescent tracer. UDP 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 glycosyltransferase in a high throughput format.

Direct detection of UDP with a fluorescence polarization (FP) readout provides a safe, HTS-compatible alternative to cumbersome radioassay methods and is more sensitive and less subject to interference than coupled assay methods, where the UDP is converted to another product. Used in over millions of wells by pharma since 2006, Transcreener technology is the most extensively validated UDP detection method available backed by the best technical support in the industry.

Direct Detection of UDP to Measure Glycosyltransferase Enzymatic Activity

Fluorescence Polarization

Learn More About the Transcreener UDP Assay

Transcreener UDP FP Assay Tech Manual

Applications

Measure enzymatic activity of glycosyltransferases
Screen compound libraries for glycosyltransferase modulators
Quantify inhibitor potency
Inhibitor selectivity profiling
Measure drug-target residence time

Features

Direct detection of unlabeled UDP
Robust Assay Z' > 0.7 under initial velocity conditions
Far-red fluorescent readouts minimize compound interference
A safe, non-radioactive method
Use with UDP concentrations from 1 µM to 1,000 µM
Universal - use with virtually any glycosyltransferase

Easy-to-Use, Mix-&-Read, HTS Ready

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

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Example Applications

The Transcreener UDP Assay is compatible with any enzyme class that produces UDP, including UDP-glucose-, UDP-galactose-, & UDP-glucuronosyl-transferases-, as well as glycogen, hyaluranon, & cellulose synthases.

Enzyme
Toxin B
GALNT2
GALNT3
BGalT1

Far Red FP Readouts Validated on Major Multimode Readers

Supplier	Instrument	FP Assays
	TriStar²S LB 942	In Review
	Mithras² LB 943	In Review

	Cytation^TM 5	Validated
	Cytation^TM 3	Validated
	Cytation^TM 1	Validated
	Synergy^TM H1	Validated
	Synergy^TM 2/H4/4	Validated
	Synergy^TM HTX	Not Capable
	Synergy^TM Neo 2	Validated

	POLARstar^® Omega	Validated
	FLUOstar^® Omega	Not Capable
	PHERAstar^® FSX	Validated
	PHERAstar^® Plus/FS	Validated
	CLARIOstar^® /Plus	Validated
	VANTAstar^TM	Validated

	Sense	In Review

	Analyst^® GT/HT	Validated
	Gemini^® XPS/EM	Not Capable
	SpectraMax^® M2/M2e	Not Capable
	SpectraMax^® M5/M5e/FlexStation^® 3	Not Capable
	SpectraMax^® Paradigm	Validated
	SpectraMax^® iD3/iD5	Validated

	EnVision^®/EnVision^® Xcite	Validated

	Infinite^® M1000/M1000Pro/Safire2^TM	Validated
	Infinite^® M200	Not Capable
	Infinite^® F500	Validated
	Infinite^® F200/Ultra^evolution	Contact Us
	Spark^TM 10M	Validated

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 in ≤ 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 filters and settings for 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.

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What's Included


Component	Notes
UDP² Antibody	The concentration of UDP² Antibody needed for an enzyme target is dependent on the UDP-sugar concentration and buffer conditions in the enzyme reaction. Sufficient antibody is included in the kit to complete 1,000 assays (Part #3018-1K) or 10,000 assays (Part #3018-10K) to detect UDP at a concentration up to 100 µM.
UDP² Alexa Fluor^® 633 Tracer	When displaced, changes in fluorescence can be read in an FP readout. The final tracer concentration in the 20 µL reaction is 4 nM.
Stop & Detect Buffer B, 5X	The Stop & Detect Buffer B components will stop enzyme reactions that require Mg²⁺. To ensure that the enzyme reaction is stopped completely, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction. The final concentration of Stop & Detect Buffer B at the time of FP measurement is 0.25X.
UDP	Used to create the UDP-sugar/UDP standard curve.

What You Will Need


Component	Notes
Glycosyltransferase Enzyme	Transcreener^® UDP assays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphates or nucleotidases, can produce background signal and reduce the assay window.
Enzyme Buffer	User-supplied enzyme buffer components include enzyme, buffer, acceptor substrate, MgCl₂ or MnCl₂, Brij-35, and test compounds.
Assay Plates	Assay Plates Can Be Purchased Separately From BellBrook Labs. FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514.
Plate Reader	A multi-detection microplate reader configured to measure FP of the Alexa Fluor^® 633 tracer is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense a minimum volume of 2.5 µL 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.

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Transcreener UDP² Assay Kits - FP Readout

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

Cat. No.	Product Description	Price	Add to Cart
3018-1K	Transcreener UDP² FP Assay (1,000 Assay, 384 Well)	$725.00	Add to Cart
3018-10K	Transcreener UDP² FP Assay (10,000 Assay, 384 Well)	$4,099.00	Add to Cart
3018-100K	Transcreener UDP² FP Assay (10 X 10,000 Assay, 384 Well)	$20,495.00	Add to Cart

Assay Plates for FP

Cat. No.	Product Description	Price	Add to Cart
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$55.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$60.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$479.00	Add to Cart