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Recombinant E. faecalis O-Glycosidase Protein, CF

Catalog #: 8886-GH
1 Citation Datasheet / COA / SDS
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8886-GH-020
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Recombinant E. faecalis O-Glycosidase Protein, CF Summary

Product Specifications

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.
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.
Source
E. coli-derived e. faecalis O-Glycosidase protein
Glu29-Lys1324, with N-terminal Met and 6-His tag
Accession #
Q833X5
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
145 kDa
SDS-PAGE
128 kDa, reducing conditions

Product Datasheets

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8886-GH

Product Datasheet
COA
SDS

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.

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.

Assay Procedure

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
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-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; O-Glycosidase

Citation for Recombinant E. faecalis O-Glycosidase 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.

1 Citation: Showing 1 - 1

  1. Fluorescent glycan fingerprinting of SARS2 spike proteins
    Authors: ZL Wu, JM Ertelt
    Scientific Reports, 2021-10-14;11(1):20428.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay

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