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Recombinant Human MGAT4A His-tag, CF

Catalog #: 11496-GT Datasheet / COA / SDS
Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A
Catalog # Availability Size / Price Qty
11496-GT-025
Recombinant Human MGAT4A His-tag Protein Enzyme Activity Diagram.
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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human MGAT4A His-tag, CF Summary

Product Specifications

Purity
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<0.10 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to transfer N-acetyl-alpha -D-glucosamine from UDP-N-Acetyl-alpha -D-glucosamine to glycan Cy5-Fuc labeled N2f.
Able to convert >85% of the substrate glycan N2f, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human MGAT4A protein
Leu93-Asn535, with a C-terminal 6-His tag
Accession #
Q9UM21
N-terminal Sequence
Analysis
Leu93
Predicted Molecular Mass
52 kDa
SDS-PAGE
54-60 kDa, under reducing conditions.

Product Datasheets

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11496-GT

Product Datasheet
COA
SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

11496-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.

Assay Procedure

Materials
  • Assay Buffer: 50 mM Tris, 10 mM MnCl2, pH 7.0
  • Recombinant Human MGAT4A His-tag (rhMGAT4A) (Catalog # 11496-GT)
  • Cy5-Fuc labeled N2f (Cy5-N2f) (Catalog # GL304)
  • UDP-GlcNAc, 50 mM stock in 50% Ethanol
  • 17% SDS-PAGE Gel
  • Gel loading dye
  • Gel Imager with Cy5 fluorescent dye detection capability
  1. Dilute rhMGAT4A to 50 µg/mL with Assay Buffer.
  2. Create a Reaction Mix containing 0.02 µM Cy5-N2f and 1 mM UDP-GlcNAc in Assay Buffer.
  3. Combine 10 µL of rhMGAT4A and 10 µL of Reaction Mix. For a Control, combine 10 µL of Assay Buffer and 10 µL of Reaction Mix.
  4. Incubate reaction and control at 37 °C for 60 minutes.
  5. Add gel loading dye to each tube.
  6. Load half the volume of each reaction and control onto a 17% SDS-PAGE gel. Let samples migrate at least 80% down the gel before stopping.
  7. Acquire gel image and determine percent conversion of Cy5-N2f.
Per Reaction:
  • rhMGAT4A: 0.5 µg
  • Cy5-N2f: 0.2 pmol
  • UDP-GlcNAc: 0.5 mM

Scientific Data

Enzyme Activity View Larger

Recombinant Human MGAT4A His-tag Protein (Catalog # 11496-GT) catalyzes a beta 1-4GlcNAc linkage to the alpha 1-3Man arm of N-glycans.

Enzyme Activity View Larger

Recombinant Human MGAT4A His-tag Protein (Catalog # 11496-GT) activity was measured by its ability to transfer N-acetyl-alpha -D-glucosamine from UDP-N-acetyl-alpha -D-glucosamine to glycan Cy5-Fuc labeled N2f (GL304). Following treatment, a mobility shift was observed via SDS-PAGE gel.

SDS-PAGE View Larger

2 μg/lane of Recombinant Human MGAT4A His-tag Protein (Catalog # 11496-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 54-60 kDa, under reducing conditions.

Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: MGAT4A

Recombinant human Alpha-1,3-mannosyl-glycoprotein 4-beta -N-acetylglucosaminyltransferase A (MGAT4A), also known as GlcNAc-T IVa, is a metal-dependent, golgi single-pass type II membrane protein that contains an N-terminal transmembrane region, a catalytic domain, and a C-terminal carbohydrate binding module that regulates its catalytic activity (1, 2). MGAT4A is one of several human MGAT enzymes involved in the initiation and synthesis of N-glycans, with each enzyme having unique specificity and preferences (3). MGAT4 has two human isozymes that catalyze formation of the beta 1-4 GlcNAc branch in the  alpha 1-3 Mannose arm of N‑glycans (4-6). The two isozymes have different expression profiles in tissues and cancer cell lines, with MGAT4A expression within gastrointestinal tissues such as the pancreas (2, 4, 5). Loss of MGAT4A in mouse models has been shown to induce type 2 diabetes (9,10). Expression and activity of MGAT4A is particularly important during differentiation and oncogenesis (7, 8) including within choriocarcinoma, invasive mole, and placental site trophoblastic tumors (11-13). The discovery of MGAT4A modulators may lead to new therapeutics for the treatment of these diseases (10). MGAT4A may also be useful as a tool for the glycoengineering of complex N-glycans. The activity of MGAT4A was demonstrated using a fluorescent gel shift assay.

References
  1. Oka, N. et al. (2022) Glycobiology. 32:1153.
  2. Nagae, M. et. al. (2022) Commun. Biol. 5:695.
  3. Brockhausen, I. et. al. (1988) Biochemie 70:1521.
  4. Yoshida, A. et. al. (1999) Glycobiology. 9:303.
  5. Oguri, S. et. al. (2006) Glycoconj. J. 23:473.
  6. Osada, N. et. al. (2022) J. Biol. Chem. 298:102400.
  7. Nishikawa, A. et. al. (1990) Biochim. Biophys. Acta. 1035:313.
  8. Nakao, H. et. al. (1990) Biochem. Biophys. Res. Commun. 172:1260.
  9. Ohtsubo, K. et. al. (2005) Cell. 123:1307.
  10. Ohtsubo, K. et. al. (2011) Nat. Med. 17:1067.
  11. Mizuochi, T. et. al. (1983) J. Biol. Chem. 258:14126.
  12. Endo, T. et. al. (1987) Cancer Res. 47:5242.
  13. Niimi, K. et. al. (2012) Br. J. Cancer. 107:1969.
Long Name
alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A
Entrez Gene IDs
11320 (Human); 269181 (Mouse); 367252 (Rat)
Alternate Names
alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A; alpha-1,3-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; EC 2.4.1.145; GlcNAc-T IVa; GnT-4a; GNT-IV; GNT-IVA; isoenzyme A; isozyme A; mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; MGAT4A; N-Acetylglucosaminyltransferase IVa; N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase IVa; UDP-GlcNAc:a-1,3-D-mannoside b-1,4-acetylglucosaminyltransferase IV; UDP-N-acetylglucosamine: alpha-1,3-D-mannosidebeta-1,4-N-acetylglucosaminyltransferase IVa; UDP-N-acetylglucosamine:alpha1,3-d-mannosidebeta1,4-N-acetylglucosaminyltransferase

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