Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF
Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Gln22-Lys737, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
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.
|
Assay Procedure
- 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
- Dilute rBtOGA to 2 ng/μL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- 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.
- Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.
- 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).
- rBtOGA: 0.1 μg
- Substrate: 1 mM
Reconstitution Calculator
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.
- Wells, L. et al. (2001) Science 291:2376.
- Wells, L. et al. (2003) Cell. Mol. Life Sci. 60:222.
- Yang, X. et al. (2008) Nature 451:964-9.
- Love, D.C.and Hanover, J.A. (2005). Sci. STKE 312:1.
- Hart, G. W. et al. (2011) Annu. Rev. Biochem. in press.
- Martinez-Fleites, C. (2008) Nat. Struct. Mol. Biol. 15:764.
- Rao, F. V. et al. (2006) The EMBO J. 25:1569.
- Dennis, R.J. et al. (2006) Nat. Struct. Mol. Biol. 13:365.
- He, Y. et al. (2008) Carbohydr. Res. 344:627.
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.
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Citations: Showing 1 - 2
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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, 2018-02-01;0(0):.
Species: Human, Mouse
Sample Types: Whole Cells
Applications: Click Chemistry -
The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer.
Authors: Rambaruth ND, Greenwell P, Dwek MV
Glycobiology, 2012-02-09;22(6):839-48.
Species: Human
Sample Types: Protein
Applications: Enzyme Assay
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