Recombinant Human POGLUT1 Protein, CF

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

Recombinant Human POGLUT1 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze UDP-Glucose. The specific activity is >2 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Protein O-Glucosyltransferase 1/POGLUT1 protein
  Met1-Leu388, with a C-terminal 6-His tag
  Accession # Q8NBL1
• Accession #: Q8NBL1
• N-terminal Sequence: Arg24 and Thr66
• Protein/Peptide Type: Recombinant Enzymes
• Gene: POGLUT1
• Purity: >95%, 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

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 44 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: 40-60 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: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 10 mM MnCl₂, 5 mM CaCl₂, pH 7.5
  • Recombinant Human O‑Glucosyltransferase 1/POGLUT (rhPOGLUT1) (Catalog # 6437-GT)
  • Coupling Enzyme: Recombinant Human CD39L3/ENTPD3 (rhCD39L3/ENTPD3) (Catalog # 4400-EN)
  • Substrate: UDP-Glucose (Calbiochem, Catalog # 670120), 10 mM stock in 25% ethanol
  • Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute UDP-Glucose to 800 μM in Assay Buffer.
  2. Dilute rhCD39L3 to 4 μg/mL in Assay Buffer.
  3. Prepare reaction mixture by combining equal volumes of 800 μM UDP-Glucose and 4 μg/mL rhCD39L3.
  4. Dilute rhPOGLUT1 to 40 µg/mL in Assay Buffer.
  5. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock.
  6. 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.
  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 40 µg/mL rhPOGLUT1 into the plate. Include a substrate blank containing 25 µL of Assay Buffer.
  9. Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
  10. Cover the plate with a plate sealer and incubate at 37 ºC for 2 hours.
  11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  12. Add 100 µL of deionized water to all wells.
  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 Substrate Blank.

  Per Reaction:
  • rhPOGLUT1: 1 µg
  • rhCD39L3: 50 ng
  • Substrate: 200 µM

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 POGLUT1 Protein, CF

• 9630046K23Rik
• C3orf9
• CAP10-like protein, 46 kDa
• chromosome 3 open reading frame 9
• CLP46
• EC 2.4.1.-
• hCLP46CAP10-like 46 kDa protein
• KDELC family like 1
• KDELCL1
• KTEL (Lys-Tyr-Glu-Leu) containing 1
• KTEL motif-containing protein 1
• KTELC1
• MDS010
• MDSRP
• MDSRPKTELC1
• MGC32995
• Myelodysplastic syndromes relative protein
• POGLUT1
• protein O-glucosyltransferase 1
• Rumi
• x 010 protein

Background

O-Glucosyltransferase1 (POGLUT1) is a homologue of Rumi from Drosophila, an endoplasmic reticulum (ER)-retaining glucosyltransferase, that adds glucose to serine residues within the consensus sequence of C1‑X‑S‑X‑P‑C2 in Notch EGF repeats, thereby regulating cell-fate decisions (1). It is also known as CAP10-like protein (2) and KTELC1 due to the highly conserved CAP10 domain and the presence of an ER-retaining motif, KTEL, at the C‑terminus. The human gene is reported to be involved in the pathogenesis of both acute myelogenous and T‑acute lymphoblastic leukemias (3). Recently, POGLUT1 has been demonstrated in a phosphatase-coupled glycosyltransferase assay to have hydrolase activity on UDP-Glc (4).

1. Acar, M. et al. (2008) Cell 132:247.
2. Teng, Y. et al. (2006) Gene 371:7
3. Wang, Y. et al. (2010) Genet Test Mol Biomarkers 14:127.
4. Wu, Z.L. et al. (2010) Glycobiology, in press.

Bioinformatics

• Gene Symbol: POGLUT1
• Uniprot:
  • Q8NBL1()