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

Catalog # Availability Size / Price Qty
6779-GH-020
R&D Systems Recombinant Proteins and Enzymes
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Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF Summary

Learn more about Fluorescent Glycan Labeling and Detection

Product Specifications

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.
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.
Source
E. coli-derived b. thetaiotaomicron O-GlcNAcase/OGA protein
Gln22-Lys737, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
83 kDa
SDS-PAGE
66-76 kDa, reducing conditions

Product Datasheets

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6779-GH

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

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.

Assay Procedure

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
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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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.
Entrez Gene IDs
10724 (Human); 76055 (Mouse); 154968 (Rat); 1074035 (B. thetaiotaomicron)
Alternate Names
bifunctional protein NCOAT; FLJ11229; GH84; HEXC; HEXC3; Hexosaminidase B; hyaluronidase in meningioma; KIAA0679; MEA5FLJ23355; meningioma expressed antigen 5 (hyaluronidase); Meningioma-expressed antigen 5; MGEA5; NCOAT; Nuclear cytoplasmic O-GlcNAcase and acetyltransferase; OGA; OGlcNAcase; O-GlcNAcase

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

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

2 Citations: Showing 1 - 2
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  1. Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.
    Authors: Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R
    Glycobiology, 2017;0(0):.
    Species: Human
    Sample Types: Whole Cells
    Applications: Click Chemistry
  2. The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer.
    Authors: Rambaruth ND, Greenwell P, Dwek MV
    Glycobiology, 2012;22(6):839-48.
    Species: Human
    Sample Types: Protein
    Applications: Enzyme Assay

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