Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2, CF

R&D Systems | Catalog # 10711-GT

HA-tag
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Key Product Details

  • R&D Systems HEK293-derived Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2 (10711-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

HEK293

Accession Number

Q10469.1

Applications

Enzyme Activity
View all N-Acetylglucosaminyltransferase 2/MGAT2 Proteins and Enzymes »

Product Specifications

Source

Human embryonic kidney cell, HEK293-derived human N-Acetylglucosaminyltransferase 2/MGAT2 protein
Asn34-Gln447, with N-terminal HA tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

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

N-terminal Sequence Analysis

Tyr of HA tag

Predicted Molecular Mass

48.7 kDa

SDS-PAGE

46-54 kDa, under reducing conditions.

Activity

Measured by its ability to modify the glycan Cy5-labeled M1N1f and thereby creating a band shift.
Able to convert >50% of the substrate glycan M1N1f to the product glycan, as measured under the described conditions.

Scientific Data Images for Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2, CF

Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2 Protein Enzyme Activity Diagram.

MGAT2 (Catalog # 10711-GT) recognizes an unmodified GlcNAc residue installed by MGAT1 (8334-GT) in the alpha 3 arm of the trimannose core of N-glycans and transfers a second GlcNAc residue to the alpha 6 arm of the structure to generate product glycan M2. MGAT2 can tolerate core-6 fucose and its Cy5 derivatives.

Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2 Protein SDS-PAGE

1 μg/lane of Recombinant Human MGAT2 His-tag (Catalog # 10711-GT) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing bands at 46-54 kDa.

Fluorescent gel mobility shift caused by rhMGAT2.

Lane 1 contained substrate glycan M1N1f' (GL302). In the presence of rhMGAT2, the glycan was modified and a mobility shift was observed.

Formulation, Preparation, and Storage

10711-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 2/MGAT2

Mannosylglycoprotein N-acetyl-glucosaminyltransferase 2 (MGAT2) is a key enzyme for the synthesis of complex type N-glycan. It specifically recognizes an unmodified GlcNAc residue installed by MGAT1 in the alpha 3 arm of the trimannosy core of N-glycans and transfers a second GlcNAc residue to the alpha 6 arm of the structure, thereby initiating the conversion of oligomannose type N-glycan to the complex type N-glycan (1-3). MGAT2 is a Golgi resident type II membrane protein with a typical domain structure of glycosyltransferases, containing a short N-terminal cytoplasmic domain, a hydrophobic non-cleavable signal-anchor domain, and a C-terminal catalytic domain (4). MGAT2 Mutations lead to type II carbohydrate-deficient glycoprotein syndrome (5) and abnormal brain development (6). Additionally, MGAT2 maybe involved in glycoantigen presentation and T cell activation (7). The activity of recombinant MGAT2 is demonstrated in an electrophoretic gel mobility shift assay using a fluorophore-labeled core N-glycan as the substrate.

References

  1. Moremen, K.W. and Haltiwanger, R.S. (2019) Nat. Chem. Biol. 15:853.
  2. Kadirvelraj, R. et al. (2018) Proc. Natl. Acad. Sci. USA 115:4637.
  3. Schachter, H. (1986). Biochem. Cell Biol. 64:163.
  4. Paulson, J.C. and Colley, K.J. (1989). J. Biol. Chem. 264:17615.
  5. Wang, Y. et al. (2001) Glycobiology 11:1051.
  6. Tan, J. et al. (1996) Am. J. Hum. Genet. 59:810.
  7. Ryan, S.O. et al. 2014 Glycobiology 24:262.

Long Name

Mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase

Alternate Names

CDG2A, GlcNAc-T II, GNT2

Entrez Gene IDs

4247 (Human); 217664 (Mouse)

Gene Symbol

MGAT2

UniProt

Q10469.1

Additional N-Acetylglucosaminyltransferase 2/MGAT2 Products

Product Documents for Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2, CF

For research use only

Related Research Areas

Glycobiology-related Enzymes

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Protocols

View specific protocols for Recombinant Human N-Acetylglucosaminyltransferase2/MGAT2, CF (10711-GT):

Materials
  • Assay Buffer: 25 mM Tris, 10 mM CaCl2, 10 mM MnCl2, pH 7.5
  • Recombinant Human MGAT2 (rhMGAT2) (Catalog # 10711-GT)
  • Uridine 5-diphospho-N-acetylglucosamine (UDP-GlcNAc) (Sigma, Catalog # U4375), 50 mM stock in 50% Ethanol
  • Uridine 5-diphosphogalactose disodium salt (UDP-Gal) (Sigma, Catalog # U4500), 10 mM stock in deionized water
  • Cy5-labeled M1N1f  (Catalog # GL301)
  • Recombinant Human B4GALT1 (rhB4GALT1)  (Catalog # 3609-GT)
  • 15% SDS-PAGE gel and SDS-PAGE reagents
  • Fluorescent imager
  1. Dilute rhMGAT2 to 10 µg/mL in Assay Buffer.
  2. Create Reaction Mixture A containing 0.130 µM Cy5-labeled M1N1f and 1.5 mM UDP-GlcNAc in Assay Buffer.
  3. Combine 10 µL of 10 µg/mL rhMGAT2 and 10 µL of Reaction Mixture A.  Prepare a control by combining 10 µL of Assay Buffer and 10 µL of Reaction Mixture A.
  4. Incubate the reaction and control at 37 °C for 30 minutes.
  5. After incubation, heat at 90 °C for 2 minutes.  Cool on ice and quickly spin down condensation.
  6. Create Reaction Mixture B containing 50 µg/mL rhB4GALT1 and 1.5 mM UDP-Gal in Assay Buffer.
  7. Add 10 µL of Reaction Mixture B to the reaction and control.
  8. Incubate the reaction and control at 37 °C for 20 minutes.
  9. Add 6 µL of the reducing sample buffer to reaction and control.
  10. Load 15 µL per lane of each reaction and control on a 15% SDS-PAGE gel and perform SDS-PAGE.  Let samples run down approximately 80% of the length of the gel.
  11. Visualize the gel with a fluorescent imager and calculate percent conversion.

Per Reaction:

  • rhMGAT2: 0.1 µg
  • Cy5-labeled M1N1f: 1.3 pmol
  • UDP-GlcNAc: 0.5 mM
  • rhB4GALT1: 0.5 µg
  • UDP-Gal: 0.5 mM




















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