Glycosyltransferase activity kit

Manufactured by R&D Systems

Sourced in United States

The Glycosyltransferase Activity Kit is a laboratory tool designed to measure the enzymatic activity of glycosyltransferases, a class of enzymes responsible for the transfer of sugar moieties to various substrates. The kit provides the necessary reagents and protocols to quantify the activity of these enzymes, which play crucial roles in numerous biological processes.

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Lab products found in correlation

Udp glucose, by Merck Group (2 mentions) Infinite 200 pro, by Tecan (2 mentions) Gdp l fucose, by Merck Group (2 mentions) Solanidine, by Merck Group (1 mentions) Readyprep protein extraction kit, by Bio-Rad (1 mentions) Stigmasterol, by Merck Group (1 mentions) Infinite m200 pro, by Tecan (1 mentions) Gdp fucose, by Merck Group (1 mentions) L2643, by Merck Group (1 mentions) Spectramax m5, by R&D Systems (1 mentions) Alpha lactose, by Merck Group (1 mentions) Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)

12 protocols using glycosyltransferase activity kit

Quantifying Glycosyltransferase Activity

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The SGT activity was measured by the Glycosyltransferase activity kit (R&D system/USA). The assay was carried out by detection of released inorganic phosphate using Malachite Green reagent25 (link). Total protein was isolated by the ReadyPrep™ Protein Extraction Kit (Bio-Rad) and quantified by Bradford method65 (link). For the SGT enzyme assay, phytosterols [stigmasterol (Sigma), solanidine (Sigma)] and UDP-glucose (Sigma) were used as accepter and donor substrates, respectively. The amount of phosphate removed from the acceptor molecule was calculated use via phosphate standard curve (Supplementary Fig. 5).

Singh G., Tiwari M., Singh S.P., Singh S., Trivedi P.K, & Misra P. (2016). Silencing of sterol glycosyltransferases modulates the withanolide biosynthesis and leads to compromised basal immunity of Withania somnifera. Scientific Reports, 6, 25562.

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B3GNT Glycosyltransferase Activity Assay

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B3GNT enzyme activity was measured using a glycosyltransferase activity kit (R&D systems). Five million Jurkat cells grown with or without SW were washed three times with Tris buffered saline and lysed with 300 μl of lysis buffer (10 mM Tris pH 7.5, 2 mM MnCl₂, 4 mM CaCl₂, 0.5% Triton-X, with protease inhibitors). The lysate was cleared of insoluble material by centrifugation and dialyzed overnight in an identical solution to remove cellular phosphate. Enzyme activity was measured following the instructions of the kit. Briefly, 25 μl of lysate was mixed with 25 μl of reaction mixture to give a final concentration of 6 mM N-acetyllactosamine, 5 mM UDP-GlcNAc, and 6 ng/μl coupling phosphatase. The reaction was allowed to proceed for two hours at 37°C, followed by visualization of released phosphate by a malachite green reagent as indicated in the kit. Absorbance at 620 nm was determined using a plate reader and converted using a phosphate standard run in parallel. Background was determined and subtracted by parallel reactions that lacked the specific acceptor N-acetyllactosamine but contained UDP-GlcNAc and coupling phosphatase.

Mkhikian H., Mortales C.L., Zhou R.W., Khachikyan K., Wu G., Haslam S.M., Kavarian P., Dell A, & Demetriou M. (2016). Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis. eLife, 5, e14814.

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Quantifying Glycosyltransferase Activity in HeLa Cells

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Glycosyltransferase activity of microsomes from HeLa shCTRL and shEXT1 was determined with the Glycosyltransferase Activity Kit (R&D Systems). A glycosyltransferase reaction was carried out in 50 μl of reaction buffer in a 96-well plate at room temperature for 20 min, according to the manufacturer’s instructions. The absorbance value for each well was measured at 620 nm with a microplate reader Tecan Infinite 200 PRO.

Kerselidou D., Dohai B.S., Nelson D.R., Daakour S., De Cock N., Hassoun Z.A., Kim D.K., Olivet J., El Assal D.C., Jaiswal A., Alzahmi A., Saha D., Pain C., Matthijssens F., Lemaitre P., Herfs M., Chapuis J., Ghesquiere B., Vertommen D., Kriechbaumer V., Knoops K., Lopez-Iglesias C., van Zandvoort M., Lambert J.C., Hanson J., Desmet C., Thiry M., Lauersen K.J., Vidal M., Van Vlierberghe P., Dequiedt F., Salehi-Ashtiani K, & Twizere J.C. (2021). Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells. Science Advances, 7(19), eabe8349.

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Quantifying β-1,3-Glucan Synthase Activity

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As mentioned in previous studies [10 (link), 36 (link)], the Glycosyltransferase Activity Kit (R&D System, Minneapolis, MN, USA) was used to determine the β-1, 3-glucan synthase activity of GFGLS2, rGFGLS2-classI and rGFGLS2-classIII by measuring the levels of inorganic phosphate released from UDP after the glycosylation reaction with UDP-glucose (Sigma-Aldrich, St. Louis, USA) as substrate [36 (link)]. The specific activity of β-1, 3-glucan synthase was defined as the amount of 1 pmol of phosphate released per microgram of protein (μg) per unit time (min) (pmol min⁻¹ μg⁻¹).
The substrate specificities of GFGLS2, rGFGLS2-classI and rGFGLS2-classIII were determined at 37 °C and pH 7.0 for 24 h using the reaction mixture containing 10 mM Tris–HCl (pH 7.0), 2.5 μg of the purified protein, and 5 mM of various substrates including UDP-glucose, glucose, laminaribiose (LAM2), laminarihexaose (LAM6), laminarinonaose (LAM9) (Megazyme, Wicklow, Ireland), and mixture of UDP-glucose with glucose, LAM2, LAM6, or LAM9, respectively. The Malachite reagents A and B were added to each well, and then the OD_620nm values representing the levels of inorganic phosphate released from UDP were determined using a plate reader (Tecan Infinite M200 PRO, Männedorf, Switzerland) to calculate their specific activity of β-1, 3-glucan synthase.

Yang Y.M., Fu X., Cui F.J., Sun L., Zan X.Y, & Sun W.J. (2023). Biochemical and structural characterization of a glucan synthase GFGLS2 from edible fungus Grifola frondosa to synthesize β-1, 3-glucan. Biotechnology for Biofuels and Bioproducts, 16, 163.

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Ktr4p Mannosyltransferase Activity Assay

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The transfer activity of Ktr4p was determined in vitro using the Glycosyltransferase Activity Kit from R&D Systems, following the instructions supplied by the manufacturer. In this coupled assay the leaving GDP of the mannosyltransferase reaction is further hydrolysed by the calcium-dependent nucleotidase ENTPD3/CD39L3 giving rise to free inorganic phosphate, which can be detected by the malachite green reagent. The reaction was initiated by the addition of 50 μM GDP-Mannose to 10 μM Ktr4p and 500 μM acceptor substrate (methyl-α-mannoside, α-1,2-mannobiose or mannose) in 25 mM Tris-HCl; pH 7.5, with 10 mM CaCl₂, 10 mM MnCl₂ at 20°C and stopped at different time points by the addition of the malachite green development solution. All reactions were performed in triplicate.

Possner D.D., Claesson M, & Guy J.E. (2015). Structure of the Glycosyltransferase Ktr4p from Saccharomyces cerevisiae. PLoS ONE, 10(8), e0136239.

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Quantification of FTVI Enzyme Activity

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Recombinant FTVI enzyme was produced in CHO cells by established techniques [15 (link)], using cDNA encoding amino acids 35-359 of the FTVI protein sequence; this sequence omits the cytoplasmic and transmembrane regions of FTVI, and encompasses the entire stem and catalytic domain of the enzyme. The specific activity of the purified enzyme was determined using the Glycosyltransferase Activity Kit (R&D Systems), as per the manufacturer’s instructions. Briefly, 0.1 μg of recombinant FTVI, 1 μL of ENTPD3/CD39L3 phosphatase, 15 nmol of N-acetyl-D-lactosamine (V-labs Inc), and 4 nmol of GDP-Fucose (Sigma-Aldrich) were mixed in 50 μL reaction buffer (25 mM Tris, 10 mM CaCl₂ and 10 mM MnCl₂, pH 7.5) and incubated in a 96-well plate at 37°C for 20 minutes. A second reaction that contained the same components except the recombinant FTVI was performed as a negative control. Reactions were terminated by the addition of 30 μL of Malachite Green Reagent A and 100 μL of water to each well. Color was developed by the addition of 30 μL of Malachite Green Reagent B to each well followed by gentle mixing and incubation at room temperature for 20 minutes. The plate was read at 620 nm using a multi-well plate reader. Phosphate standards were used to generate a calibration curve, and the specific activity of the FTVI enzyme was determined to be 60 pmol/min/μg.

Dykstra B., Lee J., Mortensen L.J., Yu H., Wu Z.L., Lin C.P., Rossi D.J, & Sackstein R. (2016). Glycoengineering of E-selectin ligands by intracellular versus extracellular fucosylation differentially affects osteotropism of human mesenchymal stem cells. Stem cells (Dayton, Ohio), 34(10), 2501-2511.

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Quantifying Fucosyltransferase Activities

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The fucosyltransferase activity was measured by the Glycosyltransferase activity kit (EA001, R&D system/USA). This kit utilizes the coupling phosphatase to remove inorganic phosphate quantitatively from the leaving nucleotide diphosphate (such as GDP) generated during glycosyltransferase reactions. The released inorganic phosphate is then assayed by Malachite Green phosphate detecting reagents and the fucosyltransferase activity is reflected by the rate of inorganic phosphorus production in a given amount of protein solution. The activity of FUT8 was measured by its ability to hydrolyze the donor substrate GDP-L-Fucose (sc-221696, Santa Cruz) and FUT2 by its ability to transfer fucose from GDP-L-Fucose to alpha-lactose (L2643, Sigma). The reaction conditions and detailed method was according to the manufacturer's protocol.

Zhang L., Gao Y., Zhang X., Guo M., Yang J., Cui H., Kong P., Niu X., Bi Y., Xu J., Yan T., Ma Y., Yang J., Qian Y., Wang F., Li H., Liu F., Cheng X, & Cui Y. (2020). TSTA3 facilitates esophageal squamous cell carcinoma progression through regulating fucosylation of LAMP2 and ERBB2. Theranostics, 10(24), 11339-11358.

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Phosphate Assay for Glycosyltransferase Activity

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Phosphate
assays were conducted
in a 96-well plate using reagents from a glycosyltransferase activity
kit (R&D Systems).^{20 (link)} Two microliters
of an enzyme reaction mixture was combined with 48 μL of doubly
distilled water (ddH₂O), followed by 30 μL of malachite
green reagent A, 100 μL of ddH₂O, and 30 μL
of malachite green reagent B. After a 20 min incubation, the absorbance
at 620 nm was measured on a Spectramax M5 microplate reader alongside
five standards of 0.0–1.0 mM KH₂PO₄ prepared
identically. Mevalonate pyrophosphate decarboxylase from Saccharomyces
cerevisiae, prepared as described previously,^{15 (link)} served as a positive control for the release
of free phosphate through the decarboxylation of (RS)-mevalonate pyrophosphate.

Vinokur J.M., Korman T.P., Cao Z, & Bowie J.U. (2014). Evidence of a Novel Mevalonate Pathway in Archaea. Biochemistry, 53(25), 4161-4168.

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Enzymatic Activities of FUT2 and FUT8

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The FUT2 and FUT8 enzyme activities were measured using the Glycosyltransferase activity kit (R&D system, MN, USA) according to the manufacturer’s instructions. The kit utilizes a colorimetric assay specific for phosphate-coupled glycosyltransferase reactions to assay the enzyme activities. The activity of FUT2 in the cell lysate was measured by its ability to transfer fucose from GDP-L-Fucose to alpha-lactose (Sigma-Aldrich, MO, USA). The activity of FUT8 in the cell lysate was measured by its ability to hydrolyze the donor substrate GDP-L-Fucose (Santa Cruz, CA, USA). The glycosyltransferase activity was expressed as phosphate concentration equivalents produced per minute (M P/min).

Huang H., He Y., Li Y., Gu M., Wu M, & Ji L. (2022). Eriodictyol suppresses the malignant progression of colorectal cancer by downregulating tissue specific transplantation antigen P35B (TSTA3) expression to restrain fucosylation. Bioengineered, 13(3), 5551-5563.

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Measuring GLT8D1 Glycosyltransferase Activity

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GLT8D1-transfected HEK293T cells were lysed in IP lysis buffer (Thermo Fisher Scientific). The lysates were incubated with anti-FLAG antibody-conjugated Dynabeads (Thermo Fisher Scientific) at 4°C overnight. Then, beads were washed with lysis buffer, and the pulled-down GLT8D1 proteins were eluted with 0.2 M glycine (pH 2.6) then neutralized with 1 M Tris-HCl (pH 9.0). Glycosyltransferase activity was measured using a glycosyltransferase activity kit (R&D Systems, Minneapolis, MN). Briefly, reactions were initiated by adding 10 ng/μL of purified GLT8D1 proteins to a reaction mixture including 5 mM UDP-galactose, 5 mM GlcNAc, and 2 ng/μL coupling phosphatase. Reactions were incubated at 37°C for 1 hour and then terminated by addition of Malachite Green reagent and OD620 read.

Tsai P.C., Jih K.Y., Shen T.Y., Liu Y.H., Lin K.P., Liao Y.C, & Lee Y.C. (2021). Genetic and Functional Analysis of Glycosyltransferase 8 Domain–Containing Protein 1 in Taiwanese Patients With Amyotrophic Lateral Sclerosis. Neurology: Genetics, 7(6), e627.

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