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  • O-Glycosidase & Neuraminidase Bundle

    Description

    1 set of this bundle includes 2,000,000 units of O-Glycosidase and 2,000 units of Neuraminidase.

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

    Neuraminidase is the common name for Acetyl-neuraminyl hydrolase (Sialidase). This Neuraminidase catalyzes the hydrolysis of α2-3, α2-6, and α2-8 linked N-acetyl-neuraminic acid residues from glycoproteins and oligosaccharides. 




    Product Source

    O-Glycosidase is cloned from Enterococcus faecalis and expressed in E.coli (1). Neuraminidase is cloned from Clostridium perfringens (2) and overexpressed in E. coli (3).

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Properties and Usage

    Unit Definition

    One unit of O-Glycosidase 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 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 of O-Glycosidase:
    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 is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by Morgan and Elson Assay (4). Note: Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.

    Unit Definition of Neuraminidase:
    One unit of Neuraminidase is defined as the amount of enzyme required to cleave > 95% of the terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC), in 5 minutes at 37°C in a total reaction volume of 10 µl.

    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 Temperature

    -20°C

    Heat Inactivation

    Quality Control

    Quality Assurance Statement

    • No contaminating endoglycosidase, 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.

    References

    1. Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.
    2. Roggentin, P. et al. (1988). FEBS Lett. 238, 31-34.
    3. Guan, C. unpublished observations. New England Biolabs.
    4. Morgan, W.T.J. and Elson, L.A. (1934). 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 the typical reaction conditions for O-Glycosidase?
    2. Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase?
    3. What is the difference between PNGase F, Endo H and O-Glycosidase?
    4. I tried using O-Glycosidase on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
    5. How much O-Glycosidase should I use to remove my carbohydrate under native conditions?
    6. How do I inhibit O-Glycosidase?
    7. What is a good positive control for O-Glycosidase?
    8. Do detergents inhibit O-Glycosidase?
    1. O-Glycosidase (P0733)
    2. O-Glycosidase Application Note 1 (P0733)

    Usage Guidelines & Tips