Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF

R&D Systems | Catalog # 6779-GH

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Key Product Details

  • R&D Systems E. coli-derived Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein (6779-GH)
  • 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

Q89ZI2

Applications

Enzyme Activity
View all O-GlcNAcase/OGA Proteins and Enzymes »

Product Specifications

Source

E. coli-derived b. thetaiotaomicron O-GlcNAcase/OGA protein
Gln22-Lys737, with an N-terminal Met and 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

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

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

83 kDa

SDS-PAGE

66-76 kDa, reducing conditions

Activity

Measured by its ability to hydrolyze 4-methylumbelliferyl-N-acetyl-beta -D-glucosaminide (4-MU-GlcNAc)
The specific activity is >3500 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

6779-GH
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Brij and Glycerol.
Shipping The product is shipped with polar packs. 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, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: O-GlcNAcase/OGA

The addition of the monosaccharide beta -N-acetyl-D-glucosamine to serine and threonine residues in proteins (O‑GlcNAc glycosylation) is a dynamic, intracellular, post‑translational modification that shares features with phosphorylation (1). Almost all major classes of intracellular proteins are modified with O‑GlcNAc glycosylation. O‑GlcNAc is known to regulate gene transcription, act as an energy sensor to desensitize insulin response, and coordinate phosphorylation to control protein activity (2, 3, 4, 5). In humans, O‑GlcNAc is introduced by a single O‑linked N‑acetylglucosamine transferase, OGT, and removed by a single glycosidase, OGA. Both OGT and OGA are cytosolic. Enzymes with high sequence homology to human OGA have been found in human pathogens and symbionts (6, 7, 8), where these enzymes are proposed to metabolize O‑GlcNAc in human proteins. OGA from the human gut symbiont Bacteroides thetaiotaomicron and its human counterpart are very similar in structure and function, and both enzymes operate via an unusual 'substrate-assisted' catalytic mechanism (8, 9). Recombinant B. thetaiotaomicron OGA can be used as an enzymatic tool to investigate O‑GlcNAc glycosylation.

References

  1. Wells, L. et al. (2001) Science 291:2376.
  2. Wells, L. et al. (2003) Cell. Mol. Life Sci. 60:222.
  3. Yang, X. et al. (2008) Nature 451:964-9.
  4. Love, D.C.and Hanover, J.A. (2005). Sci. STKE 312:1.
  5. Hart, G. W. et al. (2011)  Annu. Rev. Biochem. in press.
  6. Martinez-Fleites, C. (2008) Nat. Struct. Mol. Biol. 15:764.
  7. Rao, F. V. et al. (2006) The EMBO J. 25:1569.
  8. Dennis, R.J. et al. (2006) Nat. Struct. Mol. Biol. 13:365.
  9. He, Y. et al. (2008) Carbohydr. Res. 344:627.

Alternate Names

GH84, HEXC3, Hexosaminidase B, MGEA5, NCOAT, OGA, OGlcNAcase

Entrez Gene IDs

10724 (Human); 76055 (Mouse); 154968 (Rat); 1074035 (B. thetaiotaomicron)

Gene Symbol

OGA

UniProt

Q89ZI2

Additional O-GlcNAcase/OGA Products

Product Documents for Recombinant B. thetaiotaomicron O-GlcNAcase/OGA 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 B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF

For research use only

Citations for Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF

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Protocols

View specific protocols for Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF (6779-GH):

Materials
  • Assay Buffer: 50 mM MES, 100 mM NaCl, pH 5.5
  • Recombinant B. thetaiotaomicron O-GlcNAcase/OGA (rBtOGA) (Catalog # 6779-GH)
  • Substrate: 4-Methylumbelliferyl-N-acetyl-beta -D-glucosaminide (Sigma, Catalog # M2133), 50 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rBtOGA to 2 ng/μL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. Load into a plate 50 μL of 2 ng/μL rBtOGA, and start the reaction by adding 50 μL of 2 mM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 2 mM Substrate.
  4. Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Methylumbelliferone (4-MU) (Sigma, Catalog # M1381).

Per Well:
  • rBtOGA: 0.1 μg
  • Substrate: 1 mM

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