Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications EnzAct
Format
Carrier-Free

Order Details

Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 CF Summary

Details of Functionality
Measured by its ability to transfer N-Acetyl-alpha -D-glucosamine from UDP-N-Acetyl-alpha -D-glucosamine to a biantennary N-linked core pentasaccharide in a CD39L3 coupled assay. The specific activity is >100 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com.
Source
Chinese Hamster Ovary cell line, CHO-derived
Tyr31-Val533, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Tyr31
Protein/Peptide Type
Recombinant Enzymes
Gene
MGAT3
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
58 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
42-54 & 64-70 kDa, reducing conditions
Publications
Read Publication using
7359-GT in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
  • 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
  1. 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.
  2. 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.
  3. Dilute rhMGAT3 to 10 µg/mL in Buffer A.
  4. Dilute Donor Substrate to 1 mM in Buffer A.
  5. Dilute Acceptor Substrate to 1 mM in Buffer A.
  6. 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.
  7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Buffer A.
  8. Load 25 µL of the 10 µg/mL rhMGAT3 into the plate. Include a Control containing 25 µL of Buffer A.
  9. Start the reaction by adding 25 µL of Substrate Mixture to the wells, excluding the standard curve.
  10. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
  11. Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
  12. 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.
  13. Add 30 µL of the Malachite Green Reagent A to all wells.
  14. Add 50 µL of deionized water to all wells.
  15. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  16. Read plate at 620 nm (absorbance) in endpoint mode.
  17. 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 CF

  • beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase
  • EC 2.4.1.144
  • FLJ43371
  • GGNT3
  • GlcNAc-T III
  • GNT3
  • GNT-III
  • GNT-IIIMGC142278
  • mannosyl (beta-1,4-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase
  • MGAT3
  • MGC141943
  • N-acetylglucosaminyltransferase III
  • N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase III

Background

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).

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Publications for (1)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: EnzAct.


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Bioinformatics

Gene Symbol MGAT3
Entrez
Uniprot