TECHNICAL MANUAL GDP-Glo™ Glycosyltransferase Assay Instructions for Use of Products VA1090, VA1091 and VA1092

GDP-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 GDP-Glo™ Glycosyltransferase Assay(a) is a bioluminescent assay for detecting the activity of glycosyltransferases that use GDP-sugars as donor substrates and release GDP as a product. Glycosylating reactions catalyzed by glycosyltransferases (GTs) are central to many biological...

Description (continued) The GDP-Glo™ Glycosyltransferase Assay is a homogeneous, one-step-reagent-addition method to rapidly detect GDP formation in glycosyltransferase reactions. After the glycosyltransferase reaction, an equal volume of GDP Detection Reagent is added to simultaneously convert the GDP 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 GDP concentration by using a GDP standard curve. The light output is proportional to the concentration of GDP from low nM to 25µM (Figure...

Percent Signal Remaining Figure 2. Linearity and sensitivity of the GDP-Glo™ Glycosyltransferase Assay. Panel A. GDP standard curve was prepared over the indicated range of GDP concentrations in 25μl of 1X glycosyltransferase reaction buffer in a solid white 96-well plate. (Standard curve preparation is described in Section 3.B.) GDP-Glo™ Glycosyltransferase Assay was performed using 25μl of GDP Detection Reagent at room temperature as described in Section 4. Luminescence was recorded using a GloMax® 96 Microplate Luminometer (Cat.# E6501). Values represent the mean of four replicates. Panel...

Description (continued) GDP-Fucose + Acetyl Lactosamine Figure 3. Detection of the activity of various GDP-sugar-utilizing enzymes. Panel A. FUT2, (R&D Systems Cat.# 7770-GT) was titrated in 1X FUT2 reaction buffer the presence of 40µM of Ultra Pure GDP-Fucose (Cat.# VA1097) and 10mM α-Lactose (Sigma Cat.# L2643) as an acceptor substrate. Panel B. FUT3 (R&D Systems Cat.# 4950-GT) was titrated in 1X FUT3 reaction buffer in the presence of 40µM of Ultra Pure GDP-Fucose and 100mM Acetyl Lactosamine (Carbosynth Cat.# OA08244), as an acceptor substrate. Panel C. FUT7, (R&D Systems Cat.# 6409-GT) was...

Nucleotide Detection Buffer Dispense into aliquots. Nucleotide Detection Reagent GDP-Glo™ working solution Enzyme Dilution Buffer GDP-Glo™ Enzyme GDP Detection Reagent Add GDP Detection Reagent to completed GDP-generating reactions, and mix. Record luminescence. GloMax® Discover System Figure 4. Schematic representation of the GDP-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 TM505

2. Product Components and Storage Conditions GDP-Glo™ Glycosyltransferase Assay 200 assays VA1090 This system is sufficient for 200 assays performed in 96-well plates using a 25pl glycosyltransferase reaction and 25pl of GDP Detection Reagent. This system also can be used in 384-well plates using 5pl:5pl for a total of 1,000 assays. Includes: GDP, 10mM GDP-Glo™ Enzyme Enzyme Dilution Buffer Nucleotide Detection Buffer ATP Detection Substrate (lyophilized) 6 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...

GDP-Glo™ Glycosyltransferase Assay 400 assays VA1091 This system is sufficient for 400 assays performed in 96-well plates using a 25pl glycosyltransferase reaction and 25pl of GDP Detection Reagent. This system also can be used in 384-well plates using 5pl:5pl for a total of 2,000 assays. Includes: GDP, 10mM GDP-Glo™ Enzyme Enzyme Dilution Buffer Nucleotide Detection Buffer ATP Detection Substrate (lyophilized) GDP-Glo™ Glycosyltransferase Assay 4,000 assays VA1092 This system is sufficient for 4,000 assays performed in 96-well plates using a 25pl glycosyltransferase reaction and...

Ultra Pure GDP-Fucose, 50mM 50pl VA1097 Ultra Pure GDP-Mannose, 100mM 50pl VA1099 PRODUCT SIZE CAT.# GDP-Glo™ Glycosyltransferase Assay (VA1090) + Ultra Pure GDP-Fucose, 50mM (VA1097) 200 assays VA1093 GDP-Glo™ Glycosyltransferase Assay (VA1091) + Ultra Pure GDP-Fucose, 50mM (VA1097) 400 assays VA1094 GDP-Glo™ Glycosyltransferase Assay (VA1090) + Ultra Pure GDP-Mannose, 100mM (VA1099) 200 assays VA1095 GDP-Glo™ Glycosyltransferase Assay (VA1091) + Ultra Pure GDP-Mannose, 100mM (VA1099) 400 assays VA1096 8 Promega Corporation ■ 2800 Woods Hollow Road ■...

Preparing for the GDP-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., fucosyltransferase or mannosyltransferase) sugar acceptor substrate luminometer capable of reading multiwell plates (e.g., GloMax® Discover System [Cat.# GM3000]) plate shaker 3.A. Preparing the GDP Detection Reagent Calculate the required volumes of each reagent needed for...

3.B. Generating a Standard Curve for GDP To estimate the amount of GDP produced in the glycosyltransferase reaction, we recommend creating a standard curve of 0–25µM GDP. The GDP standards can be prepared in a separate 96-well or 384-well plate. Once the standards are prepared, transfer the appropriate amount to the same assay plate where the glycosyltransferase reaction is being performed. We recommend assaying each GDP standard concentration in triplicate. Figure 2 shows representative data from a GDP standard curve. 1. Prepare 200µl of 25µM GDP solution in preferred 1X glycosyltransferase...