Udp glo glycosyltransferase assay

Manufactured by Promega

Sourced in United States

The UDP-Glo™ Glycosyltransferase Assay is a luminescence-based kit designed to measure the activity of glycosyltransferase enzymes. The assay quantifies the production of UDP, a byproduct of the glycosyltransferase-catalyzed reaction, using a coupled enzymatic detection system.

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Lab products found in correlation

Udp glucose, by Merck Group (2 mentions) Clariostar, by BMG Labtech (1 mentions) Synergy lx multi mode microplate reader, by Agilent Technologies (1 mentions) Udp glca, by Corning (1 mentions) Udp gal, by Promega (1 mentions) Microplate reader, by Agilent Technologies (1 mentions) Infinite f200 pro, by Tecan (1 mentions) Cmp neu5ac, by Merck Group (1 mentions) Infinite m1000 pro, by Tecan (1 mentions) Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions) Synergy neo, by Agilent Technologies (1 mentions) Sigma protease inhibitor cocktail, by Merck Group (1 mentions) Udp glcnac, by Promega (1 mentions) Ezview red anti ha affinity gel beads, by Merck Group (1 mentions) Spectramax m5, by Molecular Devices (1 mentions)

16 protocols using udp glo glycosyltransferase assay

Characterization of GalS1 Glycosyltransferase

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All activated nucleotide sugars were purchased from CarboSource, Promega or Sigma. Screening of sugar-nucleotide donor specificities in the absence of acceptor substrate was done with the UDP-Glo glycosyltransferase assay (Promega) kit^{34 (link)}. Reactions (20 μl) consisted of 100 μM individual UDP-sugars (UDP-Gal, UDP-Arap, UDP-Xyl, UDP-Glc, UDP-GalA, UDP-GlcA, UDP-GlcNAc and UDP-GalNAc) and 4 μg of purified GalS1 in 50 mM HEPES and 100 mM NaCl (pH 7) at 30 °C for 18 h. The reaction mixture (5 μl) was mixed with an equal amount of UDP-Glo reagent in a 384-well assay plate (Corning 4513) and incubated for 1 h at room temperature before measuring luminescence using a Synergy LX Multi-mode microplate reader (BioTek). A standard curve was used for quantification of the UDP produced.
The quantity of UDP formed as a by-product of the GalT reaction was determined using the UDP-Glo glycosyltransferase assay (Promega) according to the manufacturer’s instructions, using either UDP-Gal (Promega) or UDP-Arap (CarboSource) as donor substrates. Standard GalT assays (20 μl) consisted of either UDP-Gal (250 μM) or UDP-Arap (400 μM) as activated nucleotide sugar donors, galactotetraose (400 μM) as an acceptor and 5 mM manganese(II) chloride in 50 mM HEPES (pH 7.0). Reactions were allowed to proceed at 30 °C for 2 h and the amount of UDP produced was determined as described above.

Prabhakar P.K., Pereira J.H., Taujale R., Shao W., Bharadwaj V.S., Chapla D., Yang J.Y., Bomble Y.J., Moremen K.W., Kannan N., Hammel M., Adams P.D., Scheller H.V, & Urbanowicz B.R. (2023). Structural and biochemical insight into a modular β-1,4-galactan synthase in plants. Nature plants, 9(3), 486-500.

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UDP-GlcNAc 2-Epimerase Activity Assay

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An UDP Glo™ Glycosyltransferase Assay (V6961; Promega Corporation; Madison, WI, USA) was performed to determine UDP-N-acetyl-glucosamine 2-epimerase activity. For this purpose, 747 µg epimerase were incubated at 37 °C with different MGO/GO concentrations (0.5 mM, 2 mM, and 5 mM) for 1 h in a volume of 3 µL. The total volume of the reaction mixture of 52.5 µL was prepared, consisting of 5 µM UDP-GlcNAc, 0.05% BSA, and glycated epimerase in buffer. This mix was incubated for 1 h at 37 °C. Afterwards, 25 µL UDP-Detection Reagent™ were added and incubated at room temperature for 1 h. Afterwards, the luminescence was detected by ClarioStar™ (BMG Labtech GmbH, Ortenberg, Germany).

Hagenhaus V., Gorenflos López J.L., Rosenstengel R., Neu C., Hackenberger C.P., Celik A., Weinert K., Nguyen M.B., Bork K., Horstkorte R, & Gesper A. (2023). Glycation Interferes with the Activity of the Bi-Functional UDP-N-Acetylglucosamine 2-Epimerase/N-Acetyl-mannosamine Kinase (GNE). Biomolecules, 13(3), 422.

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GGT Enzymatic Activity Assay

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GGT activity was measured similarly as previously described^{12 (link)}. The assay was performed in reaction buffer (100 mM HEPES buffer pH 8.0, 150 mM NaCl) at 37 °C for 1 h with 1 μM R699 enzyme, 100 μM MnCl₂, 200 μM UDP-glucose (MilliporeSigma, St. Louis, MO), 1 mM dithiothreitol and 1.75 mM galactosyl hydroxylysine (Gal-Hyl, Cayman Chemical, Ann Arbor, MI) or 2 μM deglucosylated collagen IV. Deglucosylated collagen IV was generated using a glycosidase PGGHG as previously described^{12 (link)}. GGT activity was measured by detecting UDP production with an ATP–based luciferase assay (UDP-Glo™ Glycosyltransferase Assay, Promega, Madison, WI) according to manufacturers' instructions. Experiments were performed in triplicate from distinct samples, and an unpaired t-test was used to compare the enzymatic activity of different samples. The glucosylation of galactosyl hydroxylysine was further confirmed by mass spectrometry.

Wu W., Kim J.S., Bailey A.O., Russell W.K., Richards S.J., Chen T., Chen T., Chen Z., Liang B., Yamauchi M, & Guo H. (2022). Comparative genomic and biochemical analyses identify a collagen galactosylhydroxylysyl glucosyltransferase from Acanthamoeba polyphaga mimivirus. Scientific Reports, 12, 16806.

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Confirming Glycosyltransferase Activity of Gly

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To confirm the glycosyltransferase activity of Gly, two different in vitro assays were performed. Recombinant Gly enzyme was expressed and purified as described previously (15 (link)). For gel shift assay, 15 μl of purified His-Gly (20 μm) and 10 μl of Gtf123-dGT1-GalT2 modified rFap1 (50 μm) were added into 50 μl of reaction buffer (20 mm Tris, pH 8.0, 100 mm NaCl, and 10 mm Mg²⁺). 10 mm EDTA was used as metal ion chelator. 10 mm UDP-Glc or UDP-Gal was used as sugar donor. The mixture was incubated in test tube at 37 °C water bath for 15 h. Then sample loading buffer was added to the samples for SDS-PAGE.
For the UDP-Glo Glycosyltransferase Assay (Promega) (31 ), 5 μl of the glycosyltransferase reaction mixtures that contain 20 μm recombinant Gly enzyme, 50 μm rFap1, or rFap1-Gtf123-dGT1, or rFap1-Gtf123-dGT1-GalT2 as a substrate and 25 μm UDP-Glc, or UDP-Gal were incubated in a solid white 384-well plate. After a 1-h incubation at room temperature, 5 μl of UDP detection reagent was added to each well. After another 1-h incubation at room temperature, luminescence was recorded using a BioTek Microplate reader. The values represent the mean of three experimental replicates.

Zhu F., Zhang H., Yang T., Haslam S.M., Dell A, & Wu H. (2016). Engineering and Dissecting the Glycosylation Pathway of a Streptococcal Serine-rich Repeat Adhesin. The Journal of Biological Chemistry, 291(53), 27354-27363.

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UDP-Glo Assay for TreT Activity

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TreT activity was measured using the UDP-Glo glycosyltransferase assay
(Promega) as previously described.¹⁸ Briefly, for each experimental condition, three replicates
of TreT reaction mixture containing 1 μg TreT, 200 mM NaCl, 20 mM
MgCl₂, 10 mM glucose (or analogue), and 0.4 mM UDP-sugar (all
UDP-sugars used in the UDP-Glo assay were obtained from Promega) in 25 μL
50 mM Tris-HCl buffer at pH 7.0 were set up in a white 96-well microplate. The
reactions were incubated for 2 min at room temperature. After equilibrating to
room temperature, 25 μL UDP detection reagent were added, which coupled
UDP production to a luciferase reaction. After incubation at room temperature
for 60 min, the luminescence signal was recorded using a microplate reader
(Tecan Infinite F200 Pro). The luminescence signal was fitted to a standard
curve made from a dilution series of known UDP concentrations measured in the
same 96-well microplate. Relative light units (RLUs) given by the luminescence
reader were converted to percent enzyme activity. In all experiments, reactions
without acceptor substrate were used as negative controls.

Groenevelt J.M., Meints L.M., Stothard A.I., Poston A.W., Fiolek T.J., Finocchietti D.H., Mulholand V.M., Woodruff P.J, & Swarts B.M. (2018). Chemoenzymatic Synthesis of Trehalosamine, an Aminoglycoside Antibiotic and Precursor to Mycobacterial Imaging Probes. The Journal of organic chemistry, 83(15), 8662-8667.

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Quantifying HUVEC OGA and OGT Activities

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HUVEC OGA activity was analyzed via an OGA activity kit (BMR) as per manufacturer instructions. HUVEC OGT activity was analyzed via the UDP-Glo Glycosyltransferase Assay (Promega) as per manufacturer instructions.

Basehore S.E., Bohlman S., Weber C., Swaminathan S., Zhang Y., Jang C., Arany Z, & Clyne A.M. (2021). Laminar Flow on Endothelial Cells Suppresses eNOS o-GlcNAcylation to Promote eNOS Activity. Circulation research, 129(11), 1054-1066.

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Kinetic Analysis of UGT Enzymes

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Positive substrates that were determined via LC-MS were further screened via UDP Glo™ Glycosyltransferase Assay (Promega, Mannheim, Germany) according to the manufacturer’s instruction in order to determine the kinetic parameters of the UGTs. The enzymatic reaction was performed according to [66 (link)] with minor modifications. After starting the reaction by adding UDP-glucose, samples were incubated for 30 min at 30 °C and 400 rpm. The reaction was stopped by adding 12.5 µL 0.6 M HCl and further neutralization with 1 M TRIZMA base. Five µL of the GT reaction was pipetted to a 384 well plate [40 (link)]. The calculation of kinetic data was performed with KaleidaGraph (https://www.synergy.com/; v4.5.4; accessed on 8 September 2021).

Kurze E., Ruß V., Syam N., Effenberger I., Jonczyk R., Liao J., Song C., Hoffmann T, & Schwab W. (2021). Glucosylation of (±)-Menthol by Uridine-Diphosphate-Sugar Dependent Glucosyltransferases from Plants. Molecules, 26(18), 5511.

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GFP-dTM-hTMEM5 Glycosyltransferase Assay

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GFP-dTM-hTMEM5 (from 0 to 2500 nM) was incubated with 1 mM acceptor substrate and 50 µM UDP-Xyl donor substrate at 37°C in a reaction containing 0.1 M MES pH 6.0 and 10 mM MgCl₂ for 18 hr. Detection of free UDP after hydrolysis of the sugar-nucleotide was performed using the UDP-Glo Glycosyltransferase Assay (Promega) as described above. CMP-Neu5Ac was purchased from Sigma-Aldrich.

Praissman J.L., Willer T., Sheikh M.O., Toi A., Chitayat D., Lin Y.Y., Lee H., Stalnaker S.H., Wang S., Prabhakar P.K., Nelson S.F., Stemple D.L., Moore S.A., Moremen K.W., Campbell K.P, & Wells L. (2016). The functional O-mannose glycan on α-dystroglycan contains a phospho-ribitol primed for matriglycan addition. eLife, 5, e14473.

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Monitoring Glucosyltransferase Activity of TcdB

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To monitor the activity of the glucosyltransferase activity of TcdB, the UDP-Glo™ Glycosyltransferase Assay (V6991; Promega, USA) was used, following the manufacturer’s recommendations. The reaction was performed with UDP-glucose as co-substrate and with the GTPase Rac1 as substrate, which was recombinantly purified from Escherichia coli as described previously.^{24 (link)} In brief, all reactions were performed for 1 h at 37°C in a total volume of 15 µl of glucosylation buffer (50 mM HEPES, 100 mM KCl, 2 mM MgCl₂, 1 mM MnCl₂, 100 mg/L BSA, pH 7.5), including 200 pM TcdB, 100 µM UDP-glucose, 5 µM Rac1, and, optionally, amiodarone (30 or 300 µM) or castanospermine (10 mM). Next, 10 µl of each reaction was transferred to a 96-well half-area microplate (Greiner, #675075, Austria). Reactions were then stopped by addition of 10 µl UDP Detection Reagent, followed by shaking at 1,000 rpm for 30 s. Luminescence signals were recorded using a Tecan infinite M1000Pro plate reader (Tecan Trading AG, Switzerland) with an adjusted integration time of 750 ms.

Schumacher J., Nienhaus A., Heber S., Matylitsky J., Chaves-Olarte E., Rodríguez C., Barth H, & Papatheodorou P. (2023). Exploring the inhibitory potential of the antiarrhythmic drug amiodarone against Clostridioides difficile toxins TcdA and TcdB. Gut Microbes, 15(2), 2256695.

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Bioluminescent Assay for Glycosyltransferase

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UDP-Glo Glycosyltransferase Assay (Promega) is an in
vitro bioluminescent UDP detection assay measuring
glycosyltransferase activity based on how much UDP is generated following
cleavage of UDP glucose. UDP-Glo was used to analyze JGT activity as
previously described [29 ].

Bullard W., Kieft R, & Sabatini R. (2017). A method for the efficient and selective identification of 5-hydroxymethyluracil in genomic DNA. Biology Methods & Protocols, 2(1), bpw006.

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