Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 CF
Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 CF Summary
Tyr31-Val533, with a C-terminal 6-His tag
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Buffer A: 25 mM MES, 5 mM MnCl2, pH 6.5
- Buffer B: 100 mM Tris, 5 mM CaCl2, pH 7.5
- Recombinant Human N-Acetylglucosaminyltransferase III/MGAT3 (rhMGAT3) (Catalog # 7359-GT)
- Donor Substrate: UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
- Acceptor Substrate: Biantennary N-Linked Core Pentasaccharide (V-Labs, Catalog # M592), 10 mM stock in deionized water
- Glycosyltransferase Activity Kit (Catalog # EA001)
- 96-well Clear Plate (Costar, Catalog # 92592)
- 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 rhMGAT3 to 10 µg/mL in Buffer A.
- Dilute Donor Substrate to 1 mM in Buffer A.
- Dilute Acceptor Substrate to 1 mM in Buffer A.
- Prepare Substrate Mixture by combining 120 µL of 1 mM Donor Substrate, 120 µL of 1 mM Acceptor Substrate, and 60 µL of 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 10 µg/mL rhMGAT3 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 20 minutes.
- 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 blanks, 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 Well:
- rhMGAT-3: 0.25 µg
- Coupling Phosphatase 1: 0.1 µg
- Donor Substrate: 10 nmol
- Acceptor Substrate: 10 nmol
Background: N-Acetylglucosaminyltransferase III/MGAT3
Mannosylglycoprotein N-acetyl-glucosaminyltransferase 3 (MGAT3), also known as GnT-III, regulates the branching of N-glycans. By transferring a GlcNAc residue to the beta -linked mannose of the trimannosyl core of N-linked oligosaccharides MGAT3 produces a bisecting GlcNAc structure (1). Bisecting GlcNAc is involved in a number of biological events, including the suppression of metastasis of cancer cells through modification on integrins (2, 3, 4) and the reduction of hepatitis B virus replication in hepatoma cells (5). The effect of the bisecting GlcNAc on a cancer cell counteracts that of the beta 1,6-GlcNAc created by MGAT5 (4, 6). Although MGAT3 and MGAT5 are functionally related, there is no significant homology at their primary sequences. In addition, bisecting GlcNAc on human Ig antibodies enhances their effector functions (7). Transgenic mice over expressing MGAT3 had aberrant glycosylation on apolipoprotein B and developed fatty livers (8). The enzymatic activity of recombinant human MGAT3 was determined using a phosphatase-coupled glycosyltransferase assay (9).
- Ihara, Y. et al. (1993) J. Biochem. 113:692.
- Yoshimura, M. et al. (1995) Proc. Natl. Acad. Sci. USA 92:8754.
- Isaji T, et al. (2004) J. Biol. Chem. 279:19747.
- Sato, Y. et al. (2009) J. Biol. Chem. 284:11873.
- Miyoshi, E. et al. (1995) J. Biol. Chem. 270:28311.
- Gu, J. and Taniguchi, N. (2008) Cell Adh. Migr. 2:243.
- Stanley P. (2002) Biochim. Biophys. Acta. 1573:363.
- Ihara, Y. et al. (1998) Proc. Natl. Acad. Sci. USA 95:2526.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citation for Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 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.
1 Citation: Showing 1 - 1
Phostine PST3.1a Targets MGAT5 and Inhibits Glioblastoma Initiating Cell Invasiveness and Proliferation
Authors: Z Hassani, A Saleh, S Turpault, S Khiati, W Morelle, J Vignon, JP Hugnot, E Uro-Coste, P Legrand, M Delaforge, S Loiseau, L Clarion, M Lecouvey, JN Volle, D Virieux, JL Pirat, H Duffau, N Bakalara
Mol. Cancer Res., 2017;0(0):.
Sample Types: Protein
Applications: Enzyme Assay
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Glycosyltransferase Activity Assays
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