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  • O-Glycosidase

    Description

    O-Glycosidase, also known as Endo-α-N-Acetylgalactosaminidase, catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins.

    Substrate Specificity:


    Product Source

    Cloned from Enterococcus faecalis and expressed in E. coli (1).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Glycoprotein Denaturing Buffer10X
    NP-4010%
    G7 Reaction Buffer10X

    Advantages and Features

    Applications

    • Removal of Core 1 and Core 3 O-linked disaccharide glycans from glycoproteins

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of neuraminidase digested, non-denatured fetuin (2) in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).

    Non-Denaturing Unit Definition Assay
    Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of neuraminidase digested fetuin with 1X G7 Reaction Buffer. The reaction mix is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by the Morgan and Elson Assay (2).

    1X Glycoprotein Denaturing Buffer
    0.5% SDS
    40 mM DTT

    1X NP-40
    1% NP-40 in MilliQ-H2O

    Reaction Conditions

    1X G7 Reaction Buffer
    Incubate at 37°C

    1X G7 Reaction Buffer:
    50 mM sodium phosphate
    pH 7.5 @ 25°C

    Storage Conditions

    20 mM Tris-HCl
    50 mM NaCl
    1 mM Na2EDTA
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 10 min

    Molecular Weight

    Apparent: 147000 daltons

    Quality Control

    Quality Assurance Statement

    • No contaminating exoglycosidase or proteolytic activity could be detected.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Glycosidase Activity (TLC):
      The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide.  The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
    • Protease Activity (SDS-PAGE):

      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.

    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    Notes

    1. Since O-Glycosidase is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present time. Double digest with Endo H must have NP-40 present (NP-40 does not inhibit Endo H).
    2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
    3. It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides. A general Neuraminidase (#P0720) or the O-Glycosidase and Neuraminidase bundle (#E0540S)  is recommended.
    4. Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.

    References

    1. Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.
    2. Morgan, W.T.J. and Elson, L.A. (1994). Biochem. J.. 28, 988-995.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What are Glycosidases and their uses?
    2. What are the typical reaction conditions for O-Glycosidase?
    3. I tried using O-Glycosidase on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
    4. How much O-Glycosidase should I use to remove my carbohydrate under native conditions?
    5. How do I inhibit O-Glycosidase?

    6. What is a good O-Glycosidase substrate?
    7. Do detergents inhibit O-Glycosidase?
    8. Can Neuraminidase be used together in a digest with PNGase F and O-Glycosidase?
    9. Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase?
    10. Can I double digest PNGase F and O-Glycosidase?
    11. What is the difference between PNGase F, Endo H and O-Glycosidase?
    1. O-Glycosidase Application Note 1 (P0733)
    2. O-Glycosidase (P0733)
    3. Endo-α-N-Acetylgalactosaminidase Application Note 1

    Usage Guidelines & Tips

    Application Notes

    O-Glycosidase (P0733)

    Don’t forget to include Neuraminidase (P0720) in your reaction! It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides.

    NEB’s O-Glycosidase is the only available O-Glycosidase that catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins

    You can use this enzyme under native or denaturing conditions

    Under native conditions we recommend adding more enzyme and using longer incubation times

    Under denaturing conditions the enzyme activity is increased two-fold. However, this observation may be substrate dependent.