Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF

R&D Systems | Catalog # 8334-GT

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1 (8334-GT)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

NS0

Accession Number

P26572

Applications

Enzyme Activity
View all N-Acetylglucosaminyltransferase I/MGAT1 Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human N-Acetylglucosaminyltransferase I/MGAT1 protein
Thr30-Asn445, with C-terminal 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Thr30

Predicted Molecular Mass

48 kDa

SDS-PAGE

45-52 kDa, reducing conditions

Activity

Measured by its ability to transfer N-Acetyl-D-Glucosamine from UDP-GlcNAc to alpha 1-3, alpha 1-6-Mannotriose.
The specific activity is >30 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

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).

References

  1.  Kumar R. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9948.
  2.  Ioffe, E. and Stanley, P.  (1994) Proc. Natl. Acad. Sci. USA 91:728.
  3. Zavareh, R. et al. (2012) PLoS ONE 7:e43721.
  4. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Long Name

Mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase

Alternate Names

GGNT1, GlcNac-TI, GLCT1, GNT-1

Entrez Gene IDs

4245 (Human); 17308 (Mouse); 81519 (Rat)

Gene Symbol

MGAT1

UniProt

P26572

Additional N-Acetylglucosaminyltransferase I/MGAT1 Products

Product Documents for Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF

For research use only

Related Research Areas

Glycobiology-related Enzymes

Citations for Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF

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Protocols

View specific protocols for Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF (8334-GT):

Materials
  • 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
  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 rhMGAT1 to 20 µg/mL in Buffer A.
  4. Create Substrate Mixture containing 0.4 mM UDP-GlcNAc and 2 mM alpha 1-3, alpha 1-6 Mannotriose in Buffer A.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Buffer A.
  6. Load 25 µL of the 20 µg/mL rhMGAT1 into the plate. Include a control containing 25 µL of Buffer A.
  7. Start the reaction by adding 25 µL of Substrate Mixture to the wells, excluding the standard curve.
  8. Cover the plate with a plate sealer and incubate at 37 °C for 1 hour.
  9. Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
  10. 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.
  11. Mix and incubate for 10 minutes at room temperature.
  12. Add 30 µL of the Malachite Green Reagent A to all wells.
  13. Add 50 µL of deionized water to all wells.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. 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

FAQs

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