Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF
Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Thr30-Asn445, with C-terminal 6-His tag
Analysis
Product Datasheets
8334-GT
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
8334-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Buffer A: 25 mM MES, 10 mM MnCl2, 0.02% Brij-35, pH 6.5
- Buffer B: 100 mM Tris, 5 mM CaCl2, pH 7.5
- Recombinant Human N-Acetylglucosaminyltransferase I/MGAT1 (rhMGAT1) (Catalog # 8334-GT)
- Donor Substrate: UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
- Acceptor Substrate: alpha 1-3, alpha 1-6 Mannotriose (V-Labs, Catalog # M336), 20 mM stock in deionized water
- Glycosyltransferase Activity Kit (Catalog # EA001)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Buffer A for a 100 µM stock.
- Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Buffer A. The standard curve has a range of 0.078 to 5 nmol per well.
- Dilute rhMGAT1 to 20 µg/mL in Buffer A.
- Create Substrate Mixture containing 0.4 mM UDP-GlcNAc and 2 mM alpha 1-3, alpha 1-6 Mannotriose in Buffer A.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Buffer A.
- Load 25 µL of the 20 µg/mL rhMGAT1 into the plate. Include a control containing 25 µL of Buffer A.
- Start the reaction by adding 25 µL of Substrate Mixture to the wells, excluding the standard curve.
- Cover the plate with a plate sealer and incubate at 37 °C for 1 hour.
- Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
- Add 50 µL of 2 µg/mL Coupling Phosphatase 1 to reaction wells and controls, excluding the standard curve. Also, add 50 µL of Buffer B to the wells containing the standard curve.
- Mix and incubate for 10 minutes at room temperature.
- Add 30 µL of the Malachite Green Reagent A to all wells.
- Add 50 µL of deionized water to all wells.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Phosphate released* (nmol) x (1000 pmol/nmol) |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
Per Reaction:- rhMGAT1: 0.5 µg
- Coupling Phosphatase 1: 0.1 µg
- UDP-GlcNAc: 0.2 mM
- alpha 1-3, alpha 1-6 Mannotriose: 1 mM
Background: N-Acetylglucosaminyltransferase I/MGAT1
Mannosylglycoprotein N-acetyl-glucosaminyltransferase 1 (MGAT1), also known as GnT I, is a type II transmembrane Golgi enzyme that regulates the branching of N‑glycans. By transferring a GlcNAc residue to the alpha 3-linked mannose of the trimannosyl core of N-linked oligosaccharides, MGAT1 initiates the formation of complex and hybrid N-linked carbohydrates (1). Mice lacking MGAT1 activity die at mid-gestation, revealing an essential role for these carbohydrates (2). Branched N‑glycans on cell surface proteins bind to galectins and allow the formation of a multivalent lattice thereby enhancing cell surface residency of growth factor receptors and focal adhesion proteins. Because of its key role in N-glycan synthesis, MGAT1 is a potential target for anti-cancer therapy (3). Enzymatic activity of the recombinant human MGAT1 was determined using a phosphatase coupled glycosyltransferase assay (4).
- Kumar R. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9948.
- Ioffe, E. and Stanley, P. (1994) Proc. Natl. Acad. Sci. USA 91:728.
- Zavareh, R. et al. (2012) PLoS ONE 7:e43721.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citations for Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Fluorescent glycan fingerprinting of SARS2 spike proteins
Authors: ZL Wu, JM Ertelt
Scientific Reports, 2021-10-14;11(1):20428.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay -
Dynorphin activation of kappa opioid receptor promotes microglial polarization toward M2 phenotype via TLR4/NF-&kappaB pathway
Authors: L Liu, Y Xu, H Dai, S Tan, X Mao, Z Chen
Cell Biosci, 2020-03-17;10(0):42.
Species: Mouse
Sample Types: Whole Cells
Applications: Cell Culture -
A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors
Authors: BS Hamilton, JD Wilson, MA Shumakovic, AC Fisher, JC Brooks, A Pontes, R Naran, C Heiss, C Gao, R Kardish, J Heimburg-M, P Azadi, RD Cummings, JH Merritt, MP DeLisa
Sci Rep, 2017-11-21;7(1):15907.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay
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