TECHNICAL MANUAL UMP/CMP-Glo™ Glycosyltransferase Assay Instructions for Use of Products VA1130, VA1131 and VA1132

UMP/CMP-Glo™ Glycosyltransferase Assay All technical literature is available at: www.promega.com/protocols/ Visit the web site to verify that you are using the most current version of this Technical Manual. E-mail Promega Technical Services if you have questions on use of this system: [email protected] 1. Description The UMP/CMP-Glo™ Glycosyltransferase Assay(a) is a bioluminescent assay for detecting the activity of glycosyltransferases that use CMP-, CDP- or UDP-sugars as donor substrates and release CMP or UMP. Glycosylating reactions catalyzed by glycosyltransferases (GTs) are central...

Description (continued) The UMP/CMP-Glo™ Glycosyltransferase Assay is a homogeneous, one-step-reagent-addition method to rapidly detect UMP or CMP formation in glycosyltransferase reactions. After the glycosyltransferase reaction, an equal volume of UMP/CMP Detection Reagent is added to simultaneously convert the UMP or CMP 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 UMP or CMP concentration by using a UMP or CMP standard curve. The light output is proportional to the concentration...

The UMP/CMP-Glo™ Glycosyltransferase Assay relies on the properties of a proprietary thermostable luciferase (Ultra-Glo™ Recombinant Luciferase) that is formulated to generate a stable glow-type luminescent signal and improve performance across a wide range of assay conditions. The signal produced by the luciferase reaction initiated by adding the UMP/CMP Detection Reagent is stable for more than 3 hours (Figures 2 and 3, Panel C). This extended stability eliminates the need for a luminometer equipped with injectors and allows batch-mode processing of multiple plates. Furthermore, the combination...

UMP (µM) 50 Signal-to-Background Ratio 1,037 at 60 minutes Percent Signal Remaining Figure 2. Detection linearity and sensitivity for UMP using the UMP/CMP-Glo™ Glycosyltransferase Assay. Panel A. UMP standard curve was prepared over the indicated range of UMP concentrations in 25μl of 1X glycosyltransferase reaction buffer in a solid white 96-well plate. (Standard curve preparation is described in Section 3.B.) UMP/CMP-Glo™ Glycosyltransferase Assay was performed using 25μl of UMP/CMP Detection Reagent at room temperature as described in Section 4. Luminescence was recorded using a GloMax® 96...

CMP (µM) 50 Signal-to-Background Ratio at 60 minutes Percent Signal Remaining Figure 3. Detection linearity and sensitivity for CMP using the UMP/CMP-Glo™ Glycosyltransferase Assay. Panel A. CMP standard curve was prepared over the indicated range of CMP concentrations in 25μl of 1X glycosyltransferase reaction buffer in a solid white 96-well plate. (Standard curve preparation is described in Section 3.B.) UMP/CMP-Glo™ Glycosyltransferase Assay was performed using 25μl of UMP/CMP Detection Reagent at room temperature as described in Section 4. Luminescence was recorded using a GloMax® 96 Microplate...

Description (continued) Figure 4. Detection of the activity of various sialyltransferases. Panel A. ST6GAL1 (R&D Systems Cat.# 7620-GT) was titrated in 1X ST6GAL1 reaction buffer in the presence of 100µM of CMP-NeuAc (Sigma Cat.# C8271) and 1mM LacNAc (Dextra Cat.# GN204) as an acceptor substrate. Panel B. ST3GAL1 (R&D Systems Cat.# 6905-GT-020) was titrated in 1X ST3GAL1 reaction buffer in the presence of 200µM of CMP-NeuAc (Sigma Cat.# C8271) and 0.5mM β-1,3-galactosyl-N-acetyl galactosamine (Dextra Cat.# GN213), as an acceptor substrate. All enzyme reactions were performed in 25µl volume in...

Nucleotide Detection Buffer Dispense into aliquots. Nucleotide Detection Reagent UMP/CMP-Glo™ Enzyme UMP/CMP Detection Reagent Add UMP/CMP Detection Reagent to completed UMP- or CMP-generating reactions, and mix. Record luminescence. GloMax® Discover System Figure 5. Schematic representation of the UMP/CMP-Glo™ Glycosyltransferase Assay protocol. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 www.promega.com TM506 ·

1. Description (continued) Advantages of the UMP/CMP-Glo™ Glycosyltransferase Assay • Positive linear response in the nM to pM range: Assay signal increases linearly with increasing product formation. Uses low concentrations of nucleotide-sugars, decreasing feedback glycosyltransferase inhibition issues. • High dynamic range: High signal-to-background ratios at lower concentrations of UMP or CMP means using less enzyme during the phosphoglycosyltransferase or glycosyltransferase reactions. • High sensitivity: Detect 1.25-2.5pmol UMP/CMP with a more than twofold difference over background....

UMP/CMP-Glo™ Glycosyltransferase Assay 4,000 assays VA1132 This system is sufficient for 4,000 assays performed in 96-well plates using a 25pl glycosyltransferase reaction and 25pl of UMP/CMP Detection Reagent. This system also can be used in 384-well plates using 5pl:5pl for a total of 20,000 assays. Includes: • 1 vial ATP Detection Substrate (lyophilized) Storage Conditions: Store the UMP/CMP-Glo™ Glycosyltransferase Assay kit at less than -65°C. Alternatively, store UMP/CMP-Glo™ Enzyme at less than -65°C and the other components at -10°C to -30°C. Before use, completely thaw all components...

3. Preparing for the UMP/CMP-Glo™ Glycosyltransferase Assay Materials to Be Supplied by the User • solid white multiwell plate (do not use black plates or clear plates) • enzyme reaction buffers; used for enzyme, substrate and compound dilution • multichannel pipette or automated pipetting station • glycosyltransferase (e.g., sialyltransferase or phosphoglycosyltransferase) • sugar acceptor substrate • luminometer capable of reading multiwell plates (e.g., GloMax® Discover System [Cat.# GM3000]) • plate shaker 3.A. Preparing the UMP/CMP Detection Reagent Calculate the required volumes...