UDP-Glo™ Glycosyltransferase Assay

Description

The UDP-Glo™ Glycosyltransferase Assay is a bioluminescent assay for detecting the activity of glycosyltransferases that use UDP-sugars as donor substrates and release UDP as a product. Glycosylation reactions catalyzed by glycosyltransferases are central to many biological processes, including cell:cell interactions, cell signaling and bacterial cell wall biosynthesis. Glycosyltransferases transfer sugar from a nucleotide-glycosyl donor (e.g., UDP-Galactose, UDP-Glucose, UDP-GlcNAc, UDP-GalNAc and UDP-Glucuronic Acid) to an acceptor molecule. In a glycosyltransferase reaction, the UDP moiety is released as a product; therefore, an assay that detects UDP would be suitable for monitoring the activity of the majority of glycosyltransferases.

The UDP-Glo™ Glycosyltransferase Assay is a homogeneous, single-reagent-addition method to rapidly detect UDP formation in glycosyltransferase reactions. After the glycosyltransferase reaction, an equal volume of UDP Detection Reagent is added to simultaneously convert the UDP product to ATP and generate light in a luciferase reaction. The light generated is detected using a luminometer (Figure 1). Luminescence can be correlated to UDP concentration by using an UDP standard curve.

This assay is intended for use with purified glycosyltransferases that use UDP-sugar as a donor substrate and cannot be used with whole cells or cell extract. However, glycosyltransferases can be purified from cell extract using immunoprecipitation or affinity tag pull down then used in the UDP-Glo™ Glycosyltransferase Assay.

Note: The UDP-Glo™ Glycosyltransferase Assay kits have changed from their original component configuration. The UDP-Glo™ Solution, a component of the original kits, was replaced with 1) UDP-Glo™ Enzyme and 2) Enzyme Dilution Buffer. The technical manual (#TM413) has instructions for preparing the UDP Detection Reagent. Also, note the change in the kit storage temperature from –20°C to ≤65°C.

Features – Benefits

• Universal Assay: Use any UDP-sugar-utilizing glycosyltransferase and glycosyltransferase:substrate combination, including peptide, protein, lipid and sugar substrates.
• High Dynamic Range: High signal-to-background ratios at lower concentrations of UDP means using less enzyme during the glycosyltransferase reaction.
• High Sensitivity: Detect 0.1–0.5pmol of UDP with a more than twofold difference over background.
• Linear Response in the Nanomolar to Micromolar Range: Use low concentrations of UDP-sugar, decreasing feedback glycosyltransferase inhibition issues.
• Reliable, Reproducible Data: Routinely obtain Z´ factor values >0.7 even with low UDP production rates.
• Luminescence-Based UDP Detection: Experience less overall assay interference from chemical compounds.
• Batch Plate Processing: Highly stable luminescent signal with >80% signal remaining after 3 hours.

Applications

• Profile glycosyltransferase specificity for different sugars.
• Screen library compounds for effects on glycosyltransferase enzyme activity.
• Detect chemical compound glucuranidation during drug discovery.

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