Recombinant E. faecalis O-Glycosidase Protein, CF

R&D Systems | Catalog # 8886-GH

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Key Product Details

  • R&D Systems E. coli-derived Recombinant E. faecalis O-Glycosidase Protein (8886-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

Q833X5

Applications

Enzyme Activity
View all O-Glycosidase Proteins and Enzymes »

Product Specifications

Source

E. coli-derived e. faecalis O-Glycosidase protein
Glu29-Lys1324, with N-terminal Met and 6-His tag

Purity

>75%, 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

Met

Predicted Molecular Mass

145 kDa

SDS-PAGE

128 kDa, reducing conditions

Activity

Measured by its ability to hydrolyze p-Nitrophenyl galacto-N-bioside.
The specific activity is >12,500 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

8886-GH
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
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-Glycosidase

Enterococcus faecalis O-Glycosidase, also known as endo-alpha -N-Acetylgalactosaminidase, removes O-glycans from glycoproteins. It is broadly active on Core-1, Core-2, Core-3 and Gal-Core-2 structures, which releases the following oligosaccharides, Gal beta 1-3GalNAc, Gal beta 1-3[GlcNAc beta 1-6]GalNAc, GlcNAc beta 1-3GalNAc, Gal beta 1-3[Gal beta 1-3GlcNAc beta 1-6]GalNAc, respectively (1). The enzyme is most active on Core 1, followed by Core 3, then Core 2 structures. The enzyme also has transglycosylation activity and can transfers Core-1 and Core-2 glycans to 1-alkanols, generating alkyl-oligosaccharides. Because the O-Glycosidase is not active on sialylated O-glycans, it is necessary to treat glycoproteins concomitantly with a neuraminidase for deglycosylation purpose (2).

References

  1. Goda, H. M. et al. (2008) Biochem Biophys Res Commun 375:441.
  2. Koutsioulis, D. et al. (2008). Glycobiology 18:799.

Alternate Names

Endo-alpha-N-Acetylgalactosaminidase, OGlycosidase

UniProt

Q833X5

Additional O-Glycosidase Products

Product Documents for Recombinant E. faecalis O-Glycosidase 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 E. faecalis O-Glycosidase Protein, CF

For research use only

Citations for Recombinant E. faecalis O-Glycosidase Protein, CF

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Protocols

View specific protocols for Recombinant E. faecalis O-Glycosidase Protein, CF (8886-GH):

Materials
  • Assay Buffer: 0.1 M MES, pH 6.0
  • Recombinant E. faecalis O-Glycosidase (Catalog # 8886-GH)
  • Substrate: p-Nitrophenyl galacto-N-bioside (Sigma, Catalog # N3016), 2 mM stock in deionized water
  • NaOH, 2 M stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rE. faecalis O-Glycosidase to 1 µg/mL in Assay buffer.
  2. Dilute Substrate to 0.2 mM in Assay buffer.
  3. Load 50 µL of 1 µg/mL rE. faecalis O-Glycosidase in plate, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate.
  4. Incubate sealed plate at room temperature for 5 minutes.
  5. Prepare 0.5 M NaOH in deionized water.
  6. Add 100 µL of 0.5 M NaOH to each well to stop the reactions and develop the color.
  7. Read at 405 nm (absorbance) in endpoint mode.
  8. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Abs* (OD) x well volume (L) x 1012 pmol/mol
Inc. time (min) x epsilon ** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

*Adjusted for Substrate Blank.
**Using the extinction coefficient 18100 M-1cm-1.
***Using the path correction 0.6 cm (based on a 0.0002 L volume).

Per Reaction:

  • rE. faecalis O-Glycosidase: 0.05 µg
  • Substrate: 0.1 mM

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