Recombinant Human POMGNT1 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human POMGNT1 Protein, CF Summary

Details of Functionality
Measured by its ability to transfer N-Acetyl-D-glucosamine from UDP-GlcNAc to Methyl-alpha -D-mannopyronoside. The specific activity is >500 pmol/min/μg, as measured under the described conditions.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human POMGNT1 protein
Ser66-Thr660, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Ser66
Protein/Peptide Type
Recombinant Enzymes
Gene
POMGNT1
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
68 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
65-80 kDa, reducing conditions
Publications
Read Publication using
7414-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, -20 to -70 °C as supplied.
  • 3 months, -20 to -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
  • Assay Buffer: 25 mM MES, 5 mM MnCl2, 2.5 mM CaCl2, 0.02% Brij-35, pH 6.5
  • Recombinant Human POMGNT1 (rhPOMGNT1)  (Catalog # 7414-GT)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
  • Methyl-alpha -D-mannopyranoside (Sigma, Catalog # M6882), 1 M 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 in the kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM Stock.
  2. Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
  3. Dilute Coupling Phosphatase I to 24 µg/mL in Assay Buffer.
  4. Dilute UDP-GlcNAc to 6 mM in Assay Buffer.
  5. Prepare Reaction Mixture by combining equal volumes of 24 µg/mL Coupling Phosphatase I, 6 mM UDP-GlcNAc, and 1 M methyl‑ alpha ‑D‑mannopyranoside.
  6. Dilute rhPOMGNT1 to 2 µg/mL in Assay Buffer.
  7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  8. Load 25 µL of the 2 µg/mL rhPOMGNT1 into the plate. Include a Control containing 25 µL of Assay Buffer.
    Start the reaction by adding 25 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
  9. Cover the plate with a plate sealer and incubate at 37 ºC for 20 minutes.
  10. Add 30 µL of the Malachite Green Reagent A to all wells.  Mix briefly.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. 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:
  • rhPOMGNT1:  0.050 µg
  • Coupling Phosphatase I: 0.2 µg
  • UDP-GlcNAc: 1 mM
  • Methyl-alpha -D-mannopyranoside: 167 mM
    • Notes

      Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

      Alternate Names for Recombinant Human POMGNT1 Protein, CF

      • DKFZp761B182
      • EC 2.4.1
      • EC 2.4.1.-
      • EC 2.4.1.101
      • FLJ20277
      • GnT I.2
      • MDDGA3
      • MDDGB3
      • MDDGC3
      • MEB
      • MGAT1.2
      • MGAT1.2GNTI.2
      • POMGNT1
      • protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase
      • protein O-linked-mannose beta-1,2-N-acetylglucosaminyltransferase 1
      • UDP-GlcNAc:alpha-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I.2

      Background

      Protein O-mannose beta -1,2-N-acetylglucosaminyltransferase (POMGNT1) is a Golgi-resident type II transmembrane protein that participates in O-mannosyl glycosylation (1, 2). In particular, POMGNT1 is involved in the synthesis of the GlcNAc beta 1-2Man alpha 1-O-Ser/Thr moiety present on alpha -dystroglycan and other O‑mannosylated proteins (3). A phosphorylated O‑mannosyl glycan is identified as a laminin-binding ligand of alpha -dystroglycan (4) that functions as part of the transmembrane linkage between the extracellular matrix and the cytoskeleton in skeletal muscle. POMGNT1 is widely expressed in all tissues, with the highest levels found in skeletal muscle and the heart (1, 2). Mutations of POMGNT1 are associated with muscle-eye-brain (MEB) disease (5, 6, 7). MEB is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, cobblestone lissencephaly and cerebellar hypoplasia. The enzymatic activity of the recombinant human POMGNT1 was determined using methyl-alpha -D-mannopyranoside as the acceptor substrate with a phosphatase-coupled glycosyltransferase assay (7).
      1. Zhang, W. et al. (2002) Biochem. J. 361:153.
      2. Yoshida, A. et al. (2001) Dev. Cell 1:717.
      3. Chiba, A. et al. (1997) J. Biol. Chem. 272:2156.
      4. Yoshida-Moriguchi, T. et al. (2010) Science 327:88.
      5. Manya, H. et al. (2003) Bichem. Biophys. Res. Commun. 320:39.
      6. Taniguchi, K. et al. (2003) Hum. Mol. Genet. 12:527.
      7. Diesen, C. et al. (2004) J. Med. Genet. 41:e115.
      8. Wu, Z.L. et al. (2011) Glycobiology 21:727.

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      Publications for POMGNT1 (7414-GT)(1)

      We have publications tested in 1 confirmed species: Human.

      We have publications tested in 1 application: Bioassay.


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      Bioinformatics

      Gene Symbol POMGNT1
      Uniprot