Recombinant Human O-GlcNAcTransferase/OGT Protein, CF

R&D Systems | Catalog # 8446-GT

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human O-GlcNAcTransferase/OGT Protein (8446-GT)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

O15294

Applications

Enzyme Activity
View all O-GlcNAc Transferase/OGT Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human O-GlcNAc Transferase/OGT protein
Cys323-Glu1041, with C-terminal 6-His tag

Purity

>80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Cys323

Predicted Molecular Mass

81 kDa

SDS-PAGE

70 kDa, reducing conditions

Activity

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.

Scientific Data Images for Recombinant Human O-GlcNAcTransferase/OGT Protein, CF

Detecting O-GlcNAc site on recombinant CK2 with different fluorophore-conjugated sialic acids.

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.

O-GlcNAc Detection Using the Tandem Labeling or Direct Labeling Methods.

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 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 Enzyme Activity Diagram.

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

Formulation, Preparation, and Storage

8446-GT
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and TCEP.
Shipping The product is shipped with dry ice or equivalent. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: O-GlcNAc Transferase/OGT

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).

References

  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.

Long Name

O-Linked N-Acetylglucosamine (GlcNAc) Transferase

Alternate Names

HRNT1, OGlcNAc Transferase, OGT

Entrez Gene IDs

8473 (Human); 108155 (Mouse); 26295 (Rat)

Gene Symbol

OGT

UniProt

O15294

Additional O-GlcNAc Transferase/OGT Products

Product Documents for Recombinant Human O-GlcNAcTransferase/OGT Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human O-GlcNAcTransferase/OGT Protein, CF

For research use only

Related Research Areas

Glycobiology-related Enzymes

Citations for Recombinant Human O-GlcNAcTransferase/OGT Protein, CF

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Protocols

View specific protocols for Recombinant Human O-GlcNAcTransferase/OGT Protein, CF (8446-GT):

Materials
  • 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

FAQs

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