Recombinant Human O-GlcNAcTransferase/OGT Protein, CF

O-GlcNAc was first introduced to Recombinant Human Casein Kinase 2 alpha Protein, CF (7957-CK) and then elongated by Recombinant Human B4GalT1 Protein, CF (3609-GT) and finally labeled by Recombinant Human ST6GAL1 (aa 44-406) Protein, CF (7620-GT) with different fluorophore-conjugated sialic acids. Samples were separated on 4-20% SDS-PAGE and visualized for protein with trichloroacetic acid imaging (upper panel) and fluorescent imaging (lower panel). From lane 4 to 6, O-GlcNAcylated rhCK2 was labeled with CMP-Cy3-Neu5Ac (d) (ES402), CMP-Cy5-Neu5Ac (d) (ES302) and CMP-AlexaFluor 488-Neu5Ac (d), respectively. Cy3 and AlexaFluor 488 have additional absorbance in TCE image, therefore resulting extra band intensities on the labeled bands. More information on O-GlcNAc labeling can be found at O-GlcNAc/O-GalNAc Labeling Reagents.

Nuclear extracts of HEK293 cells were tandem labeled on O-GlcNAc using Recombinant Human B4GalT1 Protein, CF (3609-GT) and by Recombinant Human ST6GAL1 (aa 44-406) Protein, CF (7620-GT). Lane 1 contains a protein Western marker, lanes 4 and 5 contain samples that were treated with Recombinant Human O-GlcNAc Transferase/OGT Protein, CF (Catalog # 8446-GT) first and then labeled for O-GlcNAc, lane 6 contains a sample that was labeled with Recombinant Human ST6Gal1 only. The left side of the figure is the fluorescent image obtained using a FluorChem fluorescent imager and the right side of the figure is the TCE image of the gel. More information on O-GlcNAc labeling can be found at O-GlcNAc/O-GalNAc Labeling Reagents.

Recombinant Human O-GlcNAc Transferase/OGT Protein, CF (Catalog # 8446-GT) catalyzes the addition of a single N-acetylglucosamine in O-glycosidic linkage to serine or threonine residues.

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

Recombinant Human O-GlcNAcTransferase/OGT Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer GlcNAc from UDP-GclNAc to peptide OGT substrate from AnaSpec, Inc. The specific activity is >25 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human O-GlcNAc Transferase/OGT protein
  Cys323-Glu1041, with C-terminal 6-His tag
• Accession #: O15294
• N-terminal Sequence: Cys323
• Protein/Peptide Type: Recombinant Enzymes
• Gene: OGT
• Purity: >80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 81 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: 70 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and TCEP.
• Purity: >80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 10X Assay Buffer (supplied in kit): 250 mM Tris, 100 mM CaCl2, pH 7.5
  • MgCl2, 1 M stock in deionized water
  • Recombinant Human O-GlcNAc Transferase/OGT (rhOGT) (Catalog # 8446-GT)
  • OGT-Substrate (AnaSpec, Inc., Catalog # 63726), 5 mM stock in deionized water
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% Ethanol/50% deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare Assay Buffer by diluting 10X Assay Buffer to1X containing 10 mM MgCl₂ with deionized water.
  2. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Prepare reaction mixture containing 0.8 mM UDP-GlcNAc, 0.4 mM OGT-Substrate, and 4 µg/mL Coupling Phosphatase 1 in Assay Buffer.
  5. Dilute rhOGT to 40 µg/mL in Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 25 µL of 40 µg/mL rhOGT into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  8. Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
  9. Seal plate and incubate at 37 °C for 30 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 sealed plate 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:
  • rhOGT: 1 µg
  • Coupling Phosphatase 1: 0.1 µg
  • OGT-Substrate: 0.2 mM
  • 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 Human O-GlcNAcTransferase/OGT Protein, CF

• EC 2.4.1
• EC 2.4.1.255
• FLJ23071
• HRNT1
• MGC22921
• O-GlcNAc transferase p110 subunit
• O-GlcNAc transferase subunit p110
• OGlcNAc Transferase
• O-GlcNAc Transferase
• O-GLCNAC
• OGT
• O-linked N-acetylglucosamine (GlcNAc) transferase(UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase)
• O-linked N-acetylglucosamine transferase 110 kDa subunit
• UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDasubunit
• uridinediphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase

Background

The O-GlcNAc post-translational modification occurs by the addition of a single N-acetyl-glucosamine residue to serine/threonine residues of cellular proteins (1, 2). Unlike other types of glycosylation, the sugar residue is not elongated into complex oligosaccharides. In fact, O-GlcNAc shares many features with protein phosphorylation, a fundamental mechanism for intracellular communication. It is postulated that O-GlcNAc has a mutually exclusive relationship with phosphorylation (3, 4). While a variety of kinases and phosphatases are involved in phosphorylation, intracellular O-GlcNAc is introduced by a single O-GlcNAc transferase, OGT, and removed by a single glycosidase, OGA. O-GlcNAc is involved in many cellular processes, including stress responses, transcription, translation, cell signaling and cell cycle regulation and in many human diseases including diabetes, Alzheimer’s disease and cancer (5, 6). By sensing levels of UDP-GlcNAc, OGT can respond to nutrient levels of all living systems via dynamically O-GlcNAcylating a wide range of nuclear and cytoplasmic proteins. Thus, strategies to modulate OGT activity may have therapeutic value for treating diabetic complications, cancer, and other diseases (7). In molecular structure, OGT comprises two distinct regions: an N-terminal region consisting of a series of tetratricopeptide repeat (TPR) units and a multidomain catalytic region (8). The enzyme activity of recombinant mouse OGT is measured using a phosphatase-coupled assay (9).

1. Hart, G.W. et al. (2007) Nature 446:1017.
2. Love D.C. and Hanover J.A. (2005) Sci. STKE. 2005:re13.
3. Comer, F.I. and Hart, G.W. (2001) Biochemistry 40:7845.
4. Hart, G.W. et al. (2011) Annu. Rev. Biochem. 80:825.
5. Ma J. and Hart, GW. (2013) Expert Rev. Proteomics 10:365.
6. Slawson C, et al. (2006) J. Biol. Chem. 97:71.
7. Dentin, R. et al. (2008) Science 319:1402.
8. Lazarus, M.B. et al. (2011) Nature 469:564.
9. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: OGT
• Uniprot:
  • O15294()