Recombinant Mouse EOGT Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse EOGT Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer GlcNAc from UDP-GlcNAc to Recombinant Drosophila Notch EGF20 (Catalog # 8117-DN). The specific activity is >0.7 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse EOGT protein
  His23-Lys523, with C-terminal 6-His tag
• Accession #: Q8BYW9
• N-terminal Sequence: His23
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Eogt
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <0.1 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 59 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: 57-61 kDa, reducing conditions

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 and NaCl.
• 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 Tris, 10 mM MgCl₂, 10 mM CaCl₂, pH 7.5
  • Recombinant Mouse EOGT (rmEOGT) (Catalog # 8116-GT)
  • Recombinant Drosophila Notch EGF20 (Notch peptide) (Catalog # 8117-DN)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% EtOH
  • 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 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 UDP-GlcNac to 10 mM in Assay Buffer.
  4. Dilute Coupling Phosphatase I to 20 ng/µL in Assay Buffer.
  5. Prepare reaction mixture containing 0.8 mM UDP-GlcNAc, 0.2 mg/mL Notch Peptide and 4 ng/µL Coupling Phosphatase I in Assay Buffer.
  6. Dilute rmEOGT to 20 ng/µL 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 20 ng/µL rmEOGT into the plate. Include a Control containing 25 µL of Assay Buffer.
  9. Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
  10. Incubate sealed plate at 37 ºC for 4 hours.
  11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  12. Add 100 µL of deionized water to all wells. Mix briefly.
  13. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  14. Read plate at 620 nm (absorbance) in endpoint mode.
  15. 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:
  • rmEOGT: 0.5 µg
  • Coupling Phosphatase I: 0.1 µg
  • Notch Peptide: 5 µg
  • UDP-GlcNAc: 0.4 mM

Notes

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

Alternate Names for Recombinant Mouse EOGT Protein, CF

• AER61 glycosyltransferase
• AER61
• C3orf64
• chromosome 3 open reading frame 64
• EC 2.4
• EOGT
• FLJ13078
• FLJ33770
• FLJ41219
• MGC34132

Background

O-GlcNAc (O-linked beta -N-acetylglucosamine) results from the addition of a single N-acetyl-glucosamine residue to serine/threonine residues. O-GlcNAc is involved in many cellular processes such as nutrient sensing, stress response, transcription, translation, cell signaling and cell cycle regulation (1). It is also involved in many human diseases including diabetes, Alzheimer’s disease and cancer (2). O-GlcNAc shares many features with protein phosphorylation, a fundamental mechanism for intracellular communication (3, 4). The modification occurs on nuclear and cytosolic proteins (3), and has been recently discovered on extracellular proteins (5). Unlike other types of glycosylation, O-GlcNAc is not elongated into complex oligosaccharides. O-GlcNAc is introduced by a single intracellular O-GlcNAc transferase (OGT) (6) and a single extracellular O-GlcNAc transferase (EOGT) (7), and removed by O-GlcNAcase (OGA) (8). EOGT is involved in the regulation of Notch receptor (9). Mutation of EOGT results in autosomal recessive Adams-Oliver syndrome (10, 11). The enzymatic activity of recombinant mouse EOGT was determined using a phosphatase-coupled assay (12).

1. Hart, G. W. et al. (2007) Nature, 446:1017.
2. Slawson, C., et al. (2006) J. Cell Biochem. 97:71.
3. Comer, F. I. and Hart, G. W. (2001), Biochemistry 40:7845.
4. Love, D. C. and Hanover, J. A. (2005), Sci. STKE 2005: re13.
5. Sakaidani, Y. et al. (2011) Nat. Commun. 2:583.
6. Haltiwanger, R. S. et al. (1990) J. Biol. Chem. 265:2563.
7. Sakaidani, Y. et al. (2012), Biochem. Biophys. Res. Commun. 419:14.
8. Gao, Y. et al. (2001), J. Biol. Chem. 276: 9838-45.
9. Muller, R. et al. (2013) PLoS One 8:e62835.
10. Cohen, I. et al. (2013) Eur. J. Hum. Genet. Online publication ahead of print.
11. Shaheen, R. et al. (2013) Am. J. Hum. Genet. 92 :598.
12. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: Eogt
• Uniprot:
  • Q8BYW9()